Anti-MUC1 antibody-drug conjugate

ABSTRACT

The present invention pertains to novel antibody drug conjugates (ADC) comprising anti-MUC1 antibody. In particular, said ADC showed significant anti-tumor efficacy.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a Track One Continuation of U.S. patentapplication Ser. No. 17/055,323, filed on Nov. 13, 2020, which claimspriority under 37 U.S.C. § 371 to International Patent Application No.PCT/EP2019/062758, filed May 17, 2019, which claims priority to and thebenefit of European Patent Application No. 18173253.8, filed on May 18,2018. The contents of these applications are hereby incorporated byreference in their entireties.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Jan. 10, 2023, isnamed 122622-0149_Updated_SL.txt and is 39160 bytes in size.

FIELD OF THE INVENTION

The present invention pertains to the field of antibody drug conjugates(ADCs). The ADC of the present invention comprises an anti-MUC1 antibodyor a mutated anti-MUC1 antibody. An ADC with a mutated anti-MUC1antibody with increased antigen binding affinity is provided. Inparticular, asparagine 57 of the heavy chain variable region issubstituted by another amino acid in the mutated version of thehumanized antibody PankoMab. Thereby, the glycosylation site in the CDR2region is deleted and the antigen binding affinity is increased. The ADCshowed significant anti-tumor efficacy. In specific embodiments, thepresent invention is directed to the therapeutic and diagnostic use ofthis antibody drug conjugates and to methods of producing such antibodydrug conjugates.

BACKGROUND OF THE INVENTION

Antibodies against tumor-associated antigens are widely usedtherapeutics against cancers. Today, many anti-cancer antibodies areapproved for human therapy. Some of these antibodies act by blockingcertain signaling pathways which are critical for survival orproliferation of specific cancer cells. Other anti-cancer antibodiesactivate the patient's immune response against the targeted cancercells, for example by initiating antibody-dependent cellularcytotoxicity (ADCC) via natural killer cells. This mechanism is inducedby binding of the antibody's Fc part to Fc receptors on the immunecells.

An interesting and important group of antibodies are those directedagainst mucin proteins. Mucins are a family of high molecular weight,heavily glycosylated proteins produced by many epithelial tissues invertebrates. They can be subdivided into mucin proteins which aremembrane-bound due to the presence of a hydrophobic membrane-spanningdomain that favors retention in the plasma membrane, and mucins whichare secreted onto mucosal surfaces or secreted to become a component ofsaliva. The human mucin protein family consists of many family members,including membrane bound MUC1.

Increased mucin production occurs in many adenocarcinomas, includingcancer of the pancreas, lung, breast, ovary, colon, etc. Mucins are alsooverexpressed in lung diseases such as asthma, bronchitis, chronicobstructive pulmonary disease or cystic fibrosis. Two membrane mucins,MUC1 and MUC4 have been extensively studied in relation to theirpathological implication in the disease process. Moreover, mucins arealso being investigated for their potential as diagnostic markers.Several antibodies directed against mucin proteins (Clin. Cancer Res.,2011 Nov. 1; 17(21):6822-30, PLoS One, 2011 Jan. 14; 6(1):e15921), inparticular MUC1, are known in the art. However, their therapeuticefficacy could still be improved.

In view of this, there is a need in the art to provide therapeuticanti-MUC1 antibodies with improved properties.

ADCs consist of three different components (antibody, linker, anddrug/payload) that are responsible for the delivery of payloadspecifically to the targeted cells. To date, four ADCs (gemtuzumabozogamicin (Mylotarg®), inotuzumab ozogamicin (Besponsa®), Brentuximabvedotin (Adcetris®), trastuzumab emtansine (T-DM1; Kadcyla®)) havegained entry into the market. Additionally, there are more than 60 ADCsbeing developed to target a wide range of blood cancers and solidtumors. ADCs have created a new paradigm for novel cancer chemotherapy.With the specificity of monoclonal antibody and the cytotoxic capacityof small molecule drugs, ADCs promise to be a large part of the futureof precision medicine as well as combination treatment. There is hencean ongoing need for the provision of further ADCs and for means, methodsand uses regarding the treatment and/or diagnosis of diseases.

As an ADC, ADCs in which exatecan is conjugated to an antibody (e.g.anti-HER2 antibody) via linker is known (WO2014/057687, WO2015/115091).However, ADCs in which exatecan is conjugated to an anti-MUC1 antibodyare not known.

SUMMARY OF THE INVENTION

The present inventors have found that deleting the glycosylation site inthe heavy chain variable region of the anti-MUC1 antibody PankoMab didnot abolish antigen binding, but rather unexpectedly increased theantigen affinity of the antibody. This was in particular surprising asthe glycosylation site is located in the secondcomplementarity-determining region of the heavy chain variable region(CDR-H2). The CDRs are those regions of an antibody which are directlyinvolved in antigen binding and provide the contact to the epitope.Therefore, generally modifying the amino acids of a CDR is expected tobe detrimental to the antigen binding affinity. The humanized PankoMabantibody additionally comprises a glycosylation site in CDR-H2, whichcarries a large carbohydrate structure. This carbohydrate structure ispresent directly at the binding interface to the antigen and hence, wasconsidered to be involved in antigen binding. However, as demonstratedin the examples, the PankoMab variant (PM-N54Q) wherein theglycosylation site is deleted by substituting the amino acid carryingthe carbohydrate structure exhibits an increased antigen bindingaffinity. In addition, the present inventors have found that a conjugateor an antibody-drug conjugate (ADC) which comprises the PankoMab or thePankoMab variant (PM-N54Q) exhibit significant anti-tumor efficacyagainst MUC1 positive tumor and that PM-N54Q-ADC showed significantanti-tumor efficacy compared to PankoMab-ADC.

Therefore, in a first aspect, the present invention is directed to aconjugate comprising an antibody conjugated to a cytotoxic agent,wherein the antibody is capable of binding to MUC1 and comprises

-   -   (i) a heavy chain variable region comprising the        complementarity-determining regions (CDRs) CDR-H1 having the        amino acid sequence of SEQ ID NO: 1, CDR-H2 having the amino        acid sequence of SEQ ID NO: 2 and CDR-H3 having the amino acid        sequence of SEQ ID NO: 3, and    -   (ii) a light chain variable region comprising the        complementarity-determining regions (CDRs) CDR-L1 having the        amino acid sequence of SEQ ID NO: 4, CDR-L2 having the amino        acid sequence of SEQ ID NO: 5 and CDR-L3 having the amino acid        sequence of SEQ ID NO: 6.

In a second aspect, the present invention is directed to a compositioncomprising the conjugate according to the invention.

According to a third aspect, the invention provides the composition orthe conjugate according to the invention for use in medicine, inparticular in the treatment, prevention or diagnosis of cancer.

In a fourth aspect, the present invention provides a method for treatingcancer in a subject in need thereof comprising, administering to thesubject with cancer a therapeutically effective amount of the conjugateaccording to the invention.

In a fifth aspect, the present invention provides kits or devicescomprising the conjugate according to the invention and associatedmethods that are useful in the diagnosis, detecting or monitoring ofMUC1 associated disorders such as cancer.

Other objects, features, advantages and aspects of the present inventionwill become apparent to those skilled in the art from the followingdescription and appended claims. It should be understood, however, thatthe following description, appended claims, and specific examples, whichindicate preferred embodiments of the application, are given by way ofillustration only. Various changes and modifications within the spiritand scope of the disclosed invention will become readily apparent tothose skilled in the art from reading the following.

Definitions

As used herein, the following expressions are generally intended topreferably have the meanings as set forth below, except to the extentthat the context in which they are used indicates otherwise.

The expression “comprise”, as used herein, besides its literal meaningalso includes and specifically refers to the expressions “consistessentially of” and “consist of”. Thus, the expression “comprise” refersto embodiments wherein the subject-matter which “comprises” specificallylisted elements does not comprise further elements as well asembodiments wherein the subject-matter which “comprises” specificallylisted elements may and/or indeed does encompass further elements.Likewise, the expression “have” is to be understood as the expression“comprise”, also including and specifically referring to the expressions“consist essentially of” and “consist of”. The term “consist essentiallyof”, where possible, in particular refers to embodiments wherein thesubject-matter comprises 20% or less, in particular 15% or less, 10% orless or especially 5% or less further elements in addition to thespecifically listed elements of which the subject-matter consistsessentially of.

The term “antibody” in particular refers to a protein comprising atleast two heavy chains and two light chains connected by disulfidebonds. Each heavy chain is comprised of a heavy chain variable region(V_(H)) and a heavy chain constant region (C_(H)). Each light chain iscomprised of a light chain variable region (V_(L)) and a light chainconstant region (C_(L)). The heavy chain-constant region comprises threeor—in the case of antibodies of the IgM- or IgE-type—four heavychain-constant domains (C_(H1), C_(H2), C_(H3) and C_(H4)) wherein thefirst constant domain C_(H1) is adjacent to the variable region and maybe connected to the second constant domain C_(H2) by a hinge region. Thelight chain-constant region consists only of one constant domain. Thevariable regions can be further subdivided into regions ofhypervariability, termed complementarity determining regions (CDRs),interspersed with regions that are more conserved, termed frameworkregions (FR), wherein each variable region comprises three CDRs and fourFRs. The variable regions of the heavy and light chains contain abinding domain that interacts with an antigen. The heavy chain constantregions may be of any type such as γ-, δ-, α-, μ- or ε-type heavychains. Preferably, the heavy chain of the antibody is a γ-chain.Furthermore, the light chain constant region may also be of any typesuch as κ- or λ-type light chains. Preferably, the light chain of theantibody is a κ-chain. The terms “γ-(δ-, α-, μ- or ε-) type heavy chain”and “κ-(λ-) type light chain” refer to antibody heavy chains or antibodylight chains, respectively, which have constant region amino acidsequences derived from naturally occurring heavy or light chain constantregion amino acid sequences, especially human heavy or light chainconstant region amino acid sequences. In particular, the amino acidsequence of the constant domains of a γ-type (especially γ1-type) heavychain is at least 95%, especially at least 98%, identical to the aminoacid sequence of the constant domains of a human γ (especially the humanγ1) antibody heavy chain. Furthermore, the amino acid sequence of theconstant domain of a κ-type light chain is in particular at least 95%,especially at least 98%, identical to the amino acid sequence of theconstant domain of the human κ antibody light chain. The constantregions of the antibodies may mediate the binding of the immunoglobulinto host tissues or factors, including various cells of the immune system(e.g., effector cells) and the first component (C1q) of the classicalcomplement system. The antibody can be e.g. a humanized, human orchimeric antibody.

The antigen-binding portion of an antibody usually refers to full lengthor one or more fragments of an antibody that retains the ability tospecifically bind to an antigen. It has been shown that theantigen-binding function of an antibody can be performed by fragments ofa full-length antibody. Examples of binding fragments of an antibodyinclude a Fab fragment, a monovalent fragment consisting of the V_(L),V_(H), C_(L) and C_(H1) domains; a F(ab)₂ fragment, a bivalent fragmentcomprising two Fab fragments, each of which binds to the same antigen,linked by a disulfide bridge at the hinge region; a Fd fragmentconsisting of the V_(H) and C_(H1) domains; a Fv fragment consisting ofthe V_(L) and V_(H) domains of a single arm of an antibody; and a dAbfragment, which consists of a V_(H) domain.

The “Fab part” of an antibody in particular refers to a part of theantibody comprising the heavy and light chain variable regions (V_(H)and V_(L)) and the first domains of the heavy and light chain constantregions (C_(H1) and C_(L)). In cases where the antibody does notcomprise all of these regions, then the term “Fab part” only refers tothose of the regions V_(H), V_(L), C_(H1) and C_(L) which are present inthe antibody. Preferably, “Fab part” refers to that part of an antibodycorresponding to the fragment obtained by digesting a natural antibodywith papain which contains the antigen binding activity of the antibody.In particular, the Fab part of an antibody encompasses the antigenbinding site or antigen binding ability thereof. Preferably, the Fabpart comprises at least the V_(H) region of the antibody.

The “Fc part” of an antibody in particular refers to a part of theantibody comprising the heavy chain constant regions 2, 3 and—whereapplicable—4 (C_(H2), C_(H3) and C_(H4)). In particular, the Fc partcomprises two of each of these regions. In cases where the antibody doesnot comprise all of these regions, then the term “Fc part” only refersto those of the regions C_(H2), C_(H3) and C_(H4) which are present inthe antibody. Preferably, the Fc part comprises at least the C_(H2)region of the antibody. Preferably, “Fc part” refers to that part of anantibody corresponding to the fragment obtained by digesting a naturalantibody with papain which does not contain the antigen binding activityof the antibody. In particular, the Fc part of an antibody is capable ofbinding to the Fc receptor and thus, e.g. comprises an Fc receptorbinding site or an Fc receptor binding ability.

The terms “antibody” and “antibody construct”, as used herein, refer incertain embodiments to a population of antibodies or antibodyconstructs, respectively, of the same kind. In particular, allantibodies or antibody constructs of the population exhibit the featuresused for defining the antibody or antibody construct. In certainembodiments, all antibodies or antibody constructs in the populationhave the same amino acid sequence. Reference to a specific kind ofantibody, such as an antibody capable of specifically binding to MUC1,in particular refers to a population of this kind of antibody.

The term “antibody” as used herein also includes fragments andderivatives of said antibody. A “fragment or derivative” of an antibodyin particular is a protein or glycoprotein which is derived from saidantibody and is capable of binding to the same antigen, in particular tothe same epitope as the antibody. Thus, a fragment or derivative of anantibody herein generally refers to a functional fragment or derivative.In particularly preferred embodiments, the fragment or derivative of anantibody comprises a heavy chain variable region. It has been shown thatthe antigen-binding function of an antibody can be performed byfragments of a full-length antibody or derivatives thereof. Examples offragments of an antibody include (i) Fab fragments, monovalent fragmentsconsisting of the variable region and the first constant domain of eachthe heavy and the light chain; (ii) F(ab)₂ fragments, bivalent fragmentscomprising two Fab fragments linked by a disulfide bridge at the hingeregion; (iii) Fd fragments consisting of the variable region and thefirst constant domain CH1 of the heavy chain; (iv) Fv fragmentsconsisting of the heavy chain and light chain variable region of asingle arm of an antibody; (v) scFv fragments, Fv fragments consistingof a single polypeptide chain; (vi) (Fv)₂ fragments consisting of two Fvfragments covalently linked together; (vii) a heavy chain variabledomain; and (viii) multibodies consisting of a heavy chain variableregion and a light chain variable region covalently linked together insuch a manner that association of the heavy chain and light chainvariable regions can only occur intermolecular but not intramolecular.Derivatives of an antibody in particular include antibodies which bindto or compete with the same antigen as the parent antibody, but whichhave a different amino acid sequence than the parent antibody from whichit is derived. These antibody fragments and derivatives are obtainedusing conventional techniques known to those with skill in the art.

A target amino acid sequence is “derived” from or “corresponds” to areference amino acid sequence if the target amino acid sequence shares ahomology or identity over its entire length with a corresponding part ofthe reference amino acid sequence of at least 75%, more preferably atleast 80%, at least 85%, at least 90%, at least 93%, at least 95%, atleast 97%, at least 98% or at least 99%. The “corresponding part” meansthat, for example, framework region 1 of a heavy chain variable region(FRH1) of a target antibody corresponds to framework region 1 of theheavy chain variable region of the reference antibody. In particularembodiments, a target amino acid sequence which is “derived” from or“corresponds” to a reference amino acid sequence is 100% homologous, orin particular 100% identical, over its entire length with acorresponding part of the reference amino acid sequence. A “homology” or“identity” of an amino acid sequence or nucleotide sequence ispreferably determined according to the invention over the entire lengthof the reference sequence or over the entire length of the correspondingpart of the reference sequence which corresponds to the sequence whichhomology or identity is defined. An antibody derived from a parentantibody which is defined by one or more amino acid sequences, such asspecific CDR sequences or specific variable region sequences, inparticular is an antibody having amino acid sequences, such as CDRsequences or variable region sequences, which are at least 75%,preferably at least 80%, at least 85%, at least 90%, at least 93%, atleast 95%, at least 97%, at least 98% or at least 99% homologous oridentical, especially identical, to the respective amino acid sequencesof the parent antibody. In certain embodiments, the antibody derivedfrom (i.e. derivative of) a parent antibody comprises the same CDRsequences as the parent antibody, but differs in the remaining sequencesof the variable regions.

The term “antibody” as used herein also refers to multivalent andmultispecific antibodies, i.e. antibody constructs which have more thantwo binding sites each binding to the same epitope and antibodyconstructs which have one or more binding sites binding to a firstepitope and one or more binding sites binding to a second epitope, andoptionally even further binding sites binding to further epitopes.

“Specific binding” preferably means that an agent such as an antibodybinds stronger to a target such as an epitope for which it is specificcompared to the binding to another target. Examples of criteria fordetermination on whether binding is specific or not can include adissociation constant (herein referred to as “KD”). An agent bindsstronger to a first target compared to a second target if it binds tothe first target with a dissociation constant (K_(d)) which is lowerthan the dissociation constant for the second target. Preferably thedissociation constant for the target to which the agent bindsspecifically is more than 100-fold, 200-fold, 500-fold or more than1000-fold lower than the dissociation constant for the target to whichthe agent does not bind specifically. Furthermore, the term “specificbinding” in particular indicates a binding affinity between the bindingpartners with an affinity constant K_(a) of at least 10⁶ M⁻¹, preferablyat least 10⁷ M⁻¹, more preferably at least 10⁸ M⁻¹. An antibody specificfor a certain antigen in particular refers to an antibody which iscapable of binding to said antigen with an affinity having a K_(a) of atleast 10⁶ M⁻¹, preferably at least 10⁷ M⁻¹, more preferably at least 10⁸M⁻¹. For example, the term “anti-MUC1 antibody” in particular refers toan antibody specifically binding MUC1 and preferably is capable ofbinding to MUC1 with an affinity having a K_(a) of at least 10⁶ M⁻¹,preferably at least 10⁷ M⁻¹, more preferably at least 10⁸ M⁻¹.

The term “MUC1” refers to the protein MUC1, also known as mucin-1,polymorphic epithelial mucin (PEM) or cancer antigen 15-3, in particularto human MUC1 (Accession No. P15941). MUC1 is a member of the mucinfamily and encodes a membrane bound, glycosylated phosphoprotein. MUC1has a core protein mass of 120-225 kDa which increases to 250-500 kDawith glycosylation. It extends 200-500 nm beyond the surface of thecell. The protein is anchored to the apical surface of many epithelialcells by a transmembrane domain. The extracellular domain includes a 20amino acid variable number tandem repeat (VNTR) domain, with the numberof repeats varying from 20 to 120 in different individuals. Theserepeats are rich in serine, threonine and proline residues which permitsheavy O-glycosylation. In certain embodiments, the term “MUC1” refers totumor-associated MUC1 (“TA-MUC1”). TA-MUC1 is MUC1 present on cancercells. This MUC1 differs from MUC1 present on non-cancer cells in itsmuch higher expression level, its localization and its glycosylation. Inparticular, TA-MUC1 is present apolarly over the whole cell surface incancer cells, while in non-cancer cells MUC1 has a strictly apicalexpression and hence, is not accessible for systemically administeredantibodies. Furthermore, TA-MUC1 has an aberrant O-glycosylation whichexposes new peptide epitopes on the MUC1 protein backbone and newcarbohydrate tumor antigens such as the Thomsen-Friedenreich antigenalpha (TFα).

“TFα”, also called Thomsen-Friedenreich antigen alpha or Core-1, refersto the disaccharide Gal-ß1,3-GalNAc which is O-glycosidically linked inan alpha-anomeric configuration to the hydroxy amino acids serine orthreonine of proteins in carcinoma cells.

The term “sialic acid” in particular refers to any N- or O-substitutedderivatives of neuraminic acid. It may refer to both5-N-acetylneuraminic acid and 5-N-glycolylneuraminic acid, butpreferably only refers to 5-N-acetylneuraminic acid. The sialic acid, inparticular the 5-N-acetylneuraminic acid preferably is attached to acarbohydrate chain via a 2,3- or 2,6-linkage. Preferably, in theantibodies described herein both 2,3- as well as 2,6-coupled sialicacids are present.

A “relative amount of glycans” according to the invention refers to aspecific percentage or percentage range of the glycans attached to theantibodies of an antibody preparation or in a composition comprisingantibodies, respectively. In particular, the relative amount of glycansrefers to a specific percentage or percentage range of all glycanscomprised in the antibodies and thus, attached to the polypeptide chainsof the antibodies in an antibody preparation or in a compositioncomprising antibodies. 100% of the glycans refers to all glycansattached to the antibodies of the antibody preparation or in acomposition comprising antibodies, respectively. For example, a relativeamount of glycans carrying bisecting GlcNAc of 10% refers to acomposition comprising antibodies wherein 10% of all glycans comprisedin the antibodies and thus, attached to the antibody polypeptide chainsin said composition comprise a bisecting GlcNAc residue while 90% of allglycans comprised in the antibodies and thus, attached to the antibodypolypeptide chains in said composition do not comprise a bisectingGlcNAc residue. The corresponding reference amount of glycansrepresenting 100% may either be all glycan structures attached to theantibodies in the composition, or all N-glycans, i.e. all glycanstructures attached to an asparagine residue of the antibodies in thecomposition, or all complex-type glycans. The reference group of glycanstructures generally is explicitly indicated or directly derivable fromthe circumstances by the skilled person.

The term “N-glycosylation” refers to all glycans attached to asparagineresidues of the polypeptide chain of a protein. These asparagineresidues generally are part of N-glycosylation sites having the aminoacid sequence Asn-Xaa-Ser/Thr, wherein Xaa may be any amino acid exceptfor proline. Likewise, “N-glycans” are glycans attached to asparagineresidues of a polypeptide chain. The terms “glycan”, “glycan structure”,“carbohydrate”, “carbohydrate chain” and “carbohydrate structure” aregenerally used synonymously herein. N-glycans generally have a commoncore structure consisting of two N-acetylglucosamine (GlcNAc) residuesand three mannose residues, having the structureManα1,6-(Manα1,3-)Manβ1,4-GlcNAcβ1,4-GlcNAcβ1-Asn with Asn being theasparagine residue of the polypeptide chain. N-glycans are subdividedinto three different types, namely complex-type glycans, hybrid-typeglycans and high mannose-type glycans.

The numbers given herein, in particular the relative amounts of aspecific glycosylation property, are preferably to be understood asapproximate numbers. In particular, the numbers preferably may be up to10% higher and/or lower, in particular up to 9%, 8%, 7%, 6%, 5%, 4%, 3%,2% or 1% higher and/or lower.

The term “antibody drug conjugate (ADC)” or “conjugate” as used hereinin general refers to the linkage of an antibody or an antigen bindingfragment thereof with another agent, such as a chemotherapeutic agent, atoxin, an immunotherapeutic agent, an imaging probe, and the like. Thelinkage can be covalent bonds, or non-covalent interactions such asthrough electrostatic forces. Various linkers, known in the art anddescribed herein, can be employed in order to form the antibody drugconjugate. Additionally, the antibody drug conjugate can be provided inthe form of a fusion protein that may be expressed from a polynucleotideencoding the immune conjugate. As used herein, “fusion protein” refersto proteins created through the joining of two or more genes or genefragments which originally coded for separate proteins (includingpeptides and polypeptides). Translation of the fusion gene results in asingle protein with functional properties derived from each of theoriginal proteins.

In a “conjugate” two or more compounds are linked together. In certainembodiments, at least some of the properties from each compound areretained in the conjugate. Linking may be achieved by a covalent ornon-covalent bond. Preferably, the compounds of the conjugate are linkedvia a covalent bond. The different compounds of a conjugate may bedirectly bound to each other via one or more covalent bonds betweenatoms of the compounds. Alternatively, the compounds may be bound toeach other via a chemical moiety such as a linker molecule wherein thelinker is covalently attached to atoms of the compounds. If theconjugate is composed of more than two compounds, then these compoundsmay, for example, be linked in a chain conformation, one compoundattached to the next compound, or several compounds each may be attachedto one central compound.

The term “nucleic acid” includes single-stranded and double-strandednucleic acids and ribonucleic acids as well as deoxyribonucleic acids.It may comprise naturally occurring as well as synthetic nucleotides andcan be naturally or synthetically modified, for example by methylation,5′- and/or 3′-capping.

The term “expression cassette” in particular refers to a nucleic acidconstruct which is capable of enabling and regulating the expression ofa coding nucleic acid sequence introduced therein. An expressioncassette may comprise promoters, ribosome binding sites, enhancers andother control elements which regulate transcription of a gene ortranslation of an mRNA. The exact structure of expression cassette mayvary as a function of the species or cell type, but generally comprises5′-untranscribed and 5′- and 3′-untranslated sequences which areinvolved in initiation of transcription and translation, respectively,such as TATA box, capping sequence, CAAT sequence, and the like. Morespecifically, 5′-untranscribed expression control sequences comprise apromoter region which includes a promoter sequence for transcriptionalcontrol of the operatively connected nucleic acid. Expression cassettesmay also comprise enhancer sequences or upstream activator sequences.

According to the invention, the term “promoter” refers to a nucleic acidsequence which is located upstream (5′) of the nucleic acid sequencewhich is to be expressed and controls expression of the sequence byproviding a recognition and binding site for RNA-polymerases. The“promoter” may include further recognition and binding sites for furtherfactors which are involved in the regulation of transcription of a gene.A promoter may control the transcription of a prokaryotic or eukaryoticgene. Furthermore, a promoter may be “inducible”, i.e. initiatetranscription in response to an inducing agent, or may be “constitutive”if transcription is not controlled by an inducing agent. A gene which isunder the control of an inducible promoter is not expressed or onlyexpressed to a small extent if an inducing agent is absent. In thepresence of the inducing agent the gene is switched on or the level oftranscription is increased. This is mediated, in general, by binding ofa specific transcription factor.

The term “vector” is used here in its most general meaning and comprisesany intermediary vehicle for a nucleic acid which enables said nucleicacid, for example, to be introduced into prokaryotic and/or eukaryoticcells and, where appropriate, to be integrated into a genome. Vectors ofthis kind are preferably replicated and/or expressed in the cells.Vectors comprise plasmids, phagemids, bacteriophages or viral genomes.The term “plasmid” as used herein generally relates to a construct ofextrachromosomal genetic material, usually a circular DNA duplex, whichcan replicate independently of chromosomal DNA.

According to the invention, the term “host cell” relates to any cellwhich can be transformed or transfected with an exogenous nucleic acid.The term “host cells” comprises according to the invention prokaryotic(e.g. E. coli) or eukaryotic cells (e.g. mammalian cells, in particularhuman cells, yeast cells and insect cells). Particular preference isgiven to mammalian cells such as cells from humans, mice, hamsters,pigs, goats, or primates. The cells may be derived from a multiplicityof tissue types and comprise primary cells and cell lines. A nucleicacid may be present in the host cell in the form of a single copy or oftwo or more copies and, in one embodiment, is expressed in the hostcell.

The term “patient” means according to the invention a human being, anonhuman primate or another animal, in particular a mammal such as acow, horse, pig, sheep, goat, dog, cat or a rodent such as a mouse andrat. In a particularly preferred embodiment, the patient is a humanbeing.

The term “cancer” according to the invention in particular comprisesleukemias, seminomas, melanomas, carcinomas, teratomas, lymphomas,sarcomas, mesotheliomas, neuroblastomas, gliomas, rectal cancer,endometrial cancer, kidney cancer, adrenal cancer, thyroid cancer, bloodcancer, skin cancer, cancer of the brain, cervical cancer, intestinalcancer, liver cancer, colon cancer, stomach cancer, intestine cancer,head and neck cancer, gastrointestinal cancer, lymph node cancer,esophagus cancer, colorectal cancer, pancreas cancer, ear, nose andthroat (ENT) cancer, breast cancer, prostate cancer, bladder cancer,cancer of the uterus, ovarian cancer and lung cancer and the metastasesthereof. The term cancer according to the invention also comprisescancer metastases.

The term “tumor” means a group of cells or tissue that is formed bymisregulated cellular proliferation. Tumors may show partial or completelack of structural organization and functional coordination with thenormal tissue, and usually form a distinct mass of tissue, which may beeither benign or malignant.

The terms “tumor” and “cancer” are used interchangeably.

The term “metastasis” means the spread of cancer cells from its originalsite to another part of the body. The formation of metastasis is a verycomplex process and normally involves detachment of cancer cells from aprimary tumor, entering the body circulation and settling down to growwithin normal tissues elsewhere in the body. When tumor cellsmetastasize, the new tumor is called a secondary or metastatic tumor,and its cells normally resemble those in the original tumor. This means,for example, that, if breast cancer metastasizes to the lungs, thesecondary tumor is made up of abnormal breast cells, not of abnormallung cells. The tumor in the lung is then called metastatic breastcancer, not lung cancer.

The term “pharmaceutical composition” particularly refers to acomposition suitable for administering to a human or animal, i.e., acomposition containing components which are pharmaceutically acceptable.Preferably, a pharmaceutical composition comprises an active compound ora salt or prodrug thereof together with a carrier, diluent orpharmaceutical excipient such as buffer, preservative and tonicitymodifier. Numeric ranges described herein are inclusive of the numbersdefining the range. The headings provided herein are not limitations ofthe various aspects or embodiments of this invention which can be readby reference to the specification as a whole. According to oneembodiment, subject-matter described herein as comprising certain stepsin the case of methods or as comprising certain ingredients in the caseof compositions refers to subject-matter consisting of the respectivesteps or ingredients. It is preferred to select and combine preferredaspects and embodiments described herein and the specific subject-matterarising from a respective combination of preferred embodiments alsobelongs to the present disclosure.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based on the development of a variant of thehumanized anti-MUC1 antibody PankoMab wherein the glycosylation site inthe CDR-H2 is deleted (PM-N54Q). Deletion of the glycosylation site wasachieved by substituting amino acid Asn (asparagine) 57 of the heavychain variable region (namely, amino acid Nos: 57 of SEQ ID NO:11) byanother amino acid, especially Gln (glutamine). Asn 57 is the acceptoramino acid residue of the glycosylation site to which the carbohydratestructure is attached. Substituting this asparagine residue by anotherresidue abolishes glycosylation because the carbohydrate structure canonly be transferred to an asparagine residue by the enzymes of the hostcell. It was surprisingly found that deletion of the glycosylation sitein the CDR-H2 of PankoMab increased the antigen binding affinity of theantibody.

In view of these findings, the present invention provides a conjugatecomprising an antibody conjugated to a cytotoxic agent, wherein theantibody is capable of binding to MUC1 and comprises

-   -   (i) a heavy chain variable region comprising the        complementarity-determining regions (CDRs) CDR-H1 having the        amino acid sequence of SEQ ID NO: 1, CDR-H2 having the amino        acid sequence of SEQ ID NO: 2 and CDR-H3 having the amino acid        sequence of SEQ ID NO: 3, and    -   (ii) a light chain variable region comprising the        complementarity-determining regions (CDRs) CDR-L1 having the        amino acid sequence of SEQ ID NO: 4, CDR-L2 having the amino        acid sequence of SEQ ID NO: 5 and CDR-L3 having the amino acid        sequence of SEQ ID NO: 6.

Binding to MUC1

The antibody specifically binds to an epitope of MUC1. The epitope is inthe extracellular tandem repeats of MUC1. In certain embodiments, theantibody binds to MUC1 in a glycosylation-dependent manner. Inparticular, the antibody binds stronger if said tandem repeats areglycosylated at a threonine residue with N-acetyl galactosamine (Tn),sialyl α2-6 N-acetyl galactosamine (sTn), galactose ß1-3 N-acetylgalactosamine (TF) or galactose ß1-3 (sialyl α2-6) N-acetylgalactosamine (sTF), preferably with Tn or TF. Preferably, thecarbohydrate moiety is bound to the threonine residue by an a glycosidicbond. The epitope in the tandem repeat domain of MUC1 in particularcomprises the amino acid sequence PDTR (SEQ ID NO: 13) or PESR (SEQ IDNO: 14).

The binding to this epitope preferably is glycosylation dependent, asdescribed above, wherein in particular the binding is increased if thecarbohydrate moiety described above is attached to the threonine residueof the sequence PDTR or PESR (SEQ ID NOs: 13 and 14), respectively.

The epitope is a tumor-associated MUC1 epitope (TA-MUC1). A TA-MUC1epitope in particular refers to an epitope of MUC1 which is present ontumor cells but not on normal cells and/or which is only accessible byantibodies in the host's circulation when present on tumor cells but notwhen present on normal cells. In certain embodiments, the binding of theantibody to cells expressing TA-MUC1 epitope is stronger than thebinding to cells expressing normal, non-tumor MUC1. Preferably, saidbinding is at least 1.5-fold stronger, preferably at least 2-foldstronger, at least 5-fold stronger, at least 10-fold stronger or atleast 100-fold stronger. For TA-MUC1 binding, the antibody preferablyspecifically binds the glycosylated MUC1 tumor epitope such that thestrength of the bond is increased at least by a factor 2, preferably afactor of 4 or a factor of 10, most preferably a factor of 20 incomparison with the bond to the non-glycosylated peptide of identicallength and identical peptide sequence. Said binding can be assayed ordetermined by ELISA, RIA, surface plasmon resonance (hereinafter,referred to as “SPR”) analysis, or the like. Examples of equipment usedin the SPR analysis can include BIAcore™ (manufactured by GE HealthcareBio-Sciences Crop.), ProteOn™ (manufactured by Bio-Rad Laboratories,Inc.), DRX2 Biosensor (manufactured by Dynamic Biosensors GmbH),SPR-Navi™ (manufactured by BioNavis Oy Ltd.), Spreeta™ (manufactured byTexas Instruments Inc.), SPRi-PlexII™ (manufactured by Horiba, Ltd.),and Autolab SPR™ (manufactured by Metrohm). The binding of the antibodyto the antigen expressed on cell surface can be assayed by flowcytometry or the like.

Furthermore, the antibody may exhibit antigen binding properties similarto those of a reference antibody comprising a heavy chain variableregion with the amino acid sequence of SEQ ID NO: 11 or SEQ ID NO:10 anda light chain variable region with the amino acid sequence of SEQ ID NO:12. Preferably, the reference antibody is the humanized antibodyPankoMab. In particular, the antibody specifically binds to the sameantigen as the reference antibody, and preferably binds to said antigenwith a higher affinity. That is, the antibody preferably binds to theantigen with an affinity having a dissociation constant which is lowerthan that of the reference antibody, more preferably at least 10% lower,at least 20% lower, at least 30% lower or at least 50% lower. Moreover,the antibody preferably shows cross-specificity with the referenceantibody comprising a heavy chain variable region with the amino acidsequence of SEQ ID NO: 11 or SEQ ID NO:10 and a light chain variableregion with the amino acid sequence of SEQ ID NO: 12. In particular, thehumanized antibody is able to block the binding of the referenceantibody to MUC1 if present in a high enough concentration. This ispossible if the binding of the reference antibody to MUC1 is hinderedwhen the antibody is already bound to the antigen MUC1.

The Anti-MUC1 Antibody

An antibody capable of binding to MUC1 comprises a heavy chain variableregion comprising the complementarity-determining regions (CDRs) CDR-H1having the amino acid sequence of SEQ ID NO: 1, CDR-H2 having the aminoacid sequence of SEQ ID NO: 2 and CDR-H3 having the amino acid sequenceof SEQ ID NO: 3 and a light chain variable region comprising thecomplementarity-determining regions (CDRs) CDR-L1 having the amino acidsequence of SEQ ID NO: 4, CDR-L2 having the amino acid sequence of SEQID NO: 5 and CDR-L3 having the amino acid sequence of SEQ ID NO: 6.

In certain embodiments, the heavy chain variable region comprises anamino acid sequence which is at least 90% identical to the amino acidsequence of SEQ ID NO: 9. Especially, the heavy chain variable regioncomprises an amino acid sequence which is at least 95%, in particular atleast 98% identical to the amino acid sequence of SEQ ID NO: 9. In theseembodiments, the heavy chain variable region still comprises CDRs 5having the amino acid sequences of SEQ ID NOs: 1, 2 and 3. Hence, anysequence deviations to SEQ ID NO: 9 are located in the frameworkregions, but not in the CDRs. In particular, the heavy chain variableregion comprises the amino acid sequence of SEQ ID NO: 9.

In certain embodiments, CDR-H2 has the amino acid sequence of SEQ ID NO:2, wherein the amino acid at position 8 of SEQ ID NO: 2 is selected fromthe group consisting of glutamine, alanine, valine, histidine,tryptophan, tyrosine, lysine and arginine; especially glutamine,histidine, tryptophan, tyrosine, lysine and arginine. Preferably, theamino acid at position 8 of SEQ ID NO: 2 is glutamine, histidine,tryptophan, lysine or arginine, especially glutamine. In particular,CDR-H2 has the amino acid sequence of SEQ ID NO: 7.

In certain embodiments, CDR-H2 has the amino acid sequence of SEQ ID NO:8.

In specific embodiments, the heavy chain variable region comprises anamino acid sequence which is at least 90% identical to the amino acidsequence of SEQ ID NO: 10. Especially, the heavy chain variable regioncomprises an amino acid sequence which is at least 95%, in particular atleast 98% identical to the amino acid sequence of SEQ ID NO: 10. Inthese embodiments, the heavy chain variable region comprises the CDR-H1having the amino acid sequence of SEQ ID NO: 1, CDR-H2 having the aminoacid sequence of SEQ ID NO: 7 and CDR-H3 having the amino acid sequenceof SEQ ID NO: 3. Hence, any sequence deviations to SEQ ID NO: 10 arelocated in the framework regions, but not in the CDRs. In particular,the heavy chain variable region comprises the amino acid sequence of SEQID NO: 10.

In specific embodiments, the heavy chain variable region comprises anamino acid sequence which is at least 90% identical to the amino acidsequence of SEQ ID NO: 11. Especially, the heavy chain variable regioncomprises an amino acid sequence which is at least 95%, in particular atleast 98% identical to the amino acid sequence of SEQ ID NO: 11. Inthese embodiments, the heavy chain variable region comprises the CDR-H1having the amino acid sequence of SEQ ID NO: 1, CDR-H2 having the aminoacid sequence of SEQ ID NO: 8 and CDR-H3 having the amino acid sequenceof SEQ ID NO: 3. Hence, any sequence deviations to SEQ ID NO: 11 arelocated in the framework regions, but not in the CDRs. In particular,the heavy chain variable region comprises the amino acid sequence of SEQID NO: 11.

In certain embodiments, the light chain variable region comprises anamino acid sequence which is at least 90% identical to the amino acidsequence of SEQ ID NO: 12. Especially, the light chain variable regioncomprises an amino acid sequence which is at least 95%, in particular atleast 98% identical to the amino acid sequence of SEQ ID NO: 12. Inthese embodiments, the light chain variable region still comprises CDRshaving the amino acid sequences of SEQ ID NOs: 4, 5 and 6. Hence, anysequence deviations to SEQ ID NO: 12 are located in the frameworkregions, but not in the CDRs. In particular, the light chain variableregion comprises the amino acid sequence of SEQ ID NO: 12.

In specific embodiments, the heavy chain variable region has an aminoacid sequence which is at least 90% identical to the amino acid sequenceof SEQ ID NO: 9, wherein the CDRs still have the amino acid sequences ofSEQ ID NOs: 1, 2 and 3, and the light chain variable region has an aminoacid sequence which is at least 90% identical to the amino acid sequenceof SEQ ID NO: 12, wherein the CDRs still have the amino acid sequencesof SEQ ID NOs: 4, 5 and 6. In particular, the heavy chain variableregion has an amino acid sequence which is at least 95% identical to theamino acid sequence of SEQ ID NO: 9, wherein the CDRs still have theamino acid sequences of SEQ ID NOs: 1, 2 and 3, and the light chainvariable region has an amino acid sequence which is at least 95%identical to the amino acid sequence of SEQ ID NO: 12, wherein the CDRsstill have the amino acid sequences of SEQ ID NOs: 4, 5 and 6.

In specific embodiments, the heavy chain variable region has an aminoacid sequence which is at least 90% identical to the amino acid sequenceof SEQ ID NO: 10, wherein the CDRs still have the amino acid sequencesof SEQ ID NOs: 1, 7 and 3, and the light chain variable region has anamino acid sequence which is at least 90% identical to the amino acidsequence of SEQ ID NO: 12, wherein the CDRs still have the amino acidsequences of SEQ ID NOs: 4, 5 and 6. In particular, the heavy chainvariable region has an amino acid sequence which is at least 95%identical to the amino acid sequence of SEQ ID NO: 10, wherein the CDRsstill have the amino acid sequences of SEQ ID NOs: 1, 7 and 3, and thelight chain variable region has an amino acid sequence which is at least95% identical to the amino acid sequence of SEQ ID NO: 12, wherein theCDRs still have the amino acid sequences of SEQ ID NOs: 4, 5 and 6.

In specific embodiments, the heavy chain variable region has an aminoacid sequence which is at least 90% identical to the amino acid sequenceof SEQ ID NO: 11, wherein the CDRs still have the amino acid sequencesof SEQ ID NOs: 1, 8 and 3, and the light chain variable region has anamino acid sequence which is at least 90% identical to the amino acidsequence of SEQ ID NO: 12, wherein the CDRs still have the amino acidsequences of SEQ ID NOs: 4, 5 and 6. In particular, the heavy chainvariable region has an amino acid sequence which is at least 95%identical to the amino acid sequence of SEQ ID NO: 11, wherein the CDRsstill have the amino acid sequences of SEQ ID NOs: 1, 8 and 3, and thelight chain variable region has an amino acid sequence which is at least95% identical to the amino acid sequence of SEQ ID NO: 12, wherein theCDRs still have the amino acid sequences of SEQ ID NOs: 4, 5 and 6.

In specific embodiments, the heavy chain variable region comprises anamino acid sequence which is at least 90% identical to the amino acidsequence represented by amino acid Nos 20 to 136 of SEQ ID NO: 20.Especially, the heavy chain variable region comprises an amino acidsequence which is at least 95%, in particular at least 98% identical tothe amino acid sequence represented by amino acid Nos 20 to 136 of SEQID NO: 20. In these embodiments, the heavy chain variable regioncomprises the CDR-H1 having the amino acid sequence of SEQ ID NO: 1,CDR-H2 having the amino acid sequence of SEQ ID NO: 2 and CDR-H3 havingthe amino acid sequence of SEQ ID NO: 3. Hence, any sequence deviationsto the amino acid sequence represented by amino acid Nos 20 to 136 ofSEQ ID NO: 20 are located in the framework regions, but not in the CDRs.In particular, the heavy chain variable region comprises the amino acidsequence represented by amino acid Nos 20 to 136 of SEQ ID NO: 20. Incertain embodiments, the amino acid at position 76 of SEQ ID NO: 20 isselected from the group consisting of glutamine, alanine, valine,histidine, tryptophan, tyrosine, lysine and arginine; especiallyglutamine, histidine, tryptophan, tyrosine, lysine and arginine.Preferably, the amino acid at position 76 of SEQ ID NO: 20 is glutamine,histidine, tryptophan, lysine or arginine, especially glutamine. Inparticular, CDR-H2 has the amino acid sequence of SEQ ID NO: 7 and/orthe heavy chain variable region comprises the amino acid sequencerepresented by amino acid Nos 20 to 136 of SEQ ID NO: 23.

In specific embodiments, the light chain variable region comprises anamino acid sequence which is at least 90% identical to the amino acidsequence represented by amino acid Nos 21 to 133 of SEQ ID NO: 21.Especially, the light chain variable region comprises an amino acidsequence which is at least 95%, in particular at least 98% identical tothe amino acid sequence represented by amino acid Nos 21 to 133 of SEQID NO: 21. In these embodiments, the light chain variable region stillcomprises CDRs having the amino acid sequences of SEQ ID NOs: 4, 5 and6. Hence, any sequence deviations to amino acid sequence represented byamino acid Nos 21 to 133 of SEQ ID NO: 21 are located in the frameworkregions, but not in the CDRs. In particular, the light chain variableregion comprises the amino acid sequence represented by amino acid Nos21 to 133 of SEQ ID NO: 21.

In specific embodiments, the heavy chain variable region has an aminoacid sequence which is at least 90% identical to the amino acid sequencerepresented by amino acid Nos 20 to 136 of SEQ ID NO: 20, wherein theCDRs still have the amino acid sequences of SEQ ID NOs: 1, 7 and 3, andthe light chain variable region has an amino acid sequence which is atleast 90% identical to the amino acid sequence represented by amino acidNos 21 to 133 of SEQ ID NO: 21, wherein the CDRs still have the aminoacid sequences of SEQ ID NOs: 4, 5 and 6. In particular, the heavy chainvariable region has an amino acid sequence which is at least 95%identical to the amino acid sequence represented by amino acid Nos 20 to136 of SEQ ID NO: 20, wherein the CDRs still have the amino acidsequences of SEQ ID NOs: 1, 7 and 3, and the light chain variable regionhas an amino acid sequence which is at least 95% identical to the aminoacid sequence represented by amino acid Nos 21 to 133 of SEQ ID NO: 21,wherein the CDRs still have the amino acid sequences of SEQ ID NOs: 4, 5and 6.

In specific embodiments, the heavy chain comprises an amino acidsequence which is at least 90% identical to the amino acid sequence ofSEQ ID NO: 15. Especially, the heavy chain comprises an amino acidsequence which is at least 95%, in particular at least 98% identical tothe amino acid sequence of SEQ ID NO: 15. In these embodiments, theheavy chain comprises the CDR-H1 having the amino acid sequence of SEQID NO: 1, CDR-H2 having the amino acid sequence of SEQ ID NO: 2 andCDR-H3 having the amino acid sequence of SEQ ID NO: 3. Hence, anysequence deviations to SEQ ID NO: 15 are located in the frameworkregions, but not in the CDRs. In particular, the heavy chain comprisesthe amino acid sequence of SEQ ID NO: 15. In certain embodiments, theamino acid at position 57 of SEQ ID NO: 15 is selected from the groupconsisting of glutamine, alanine, valine, histidine, tryptophan,tyrosine, lysine and arginine; especially glutamine, histidine,tryptophan, tyrosine, lysine and arginine. Preferably, the amino acid atposition 57 of SEQ ID NO: 15 is glutamine, histidine, tryptophan, lysineor arginine, especially glutamine. In particular, CDR-H2 has the aminoacid sequence of SEQ ID NO: 7 and/or the heavy chain variable regioncomprises the amino acid sequence represented by amino acid Nos 20 to136 of SEQ ID NO: 22.

In specific embodiments, the heavy chain comprises an amino acidsequence which is at least 90% identical to the amino acid sequence ofSEQ ID NO: 19. Especially, the heavy chain comprises an amino acidsequence which is at least 95%, in particular at least 98% identical tothe amino acid sequence of SEQ ID NO: 19. In these embodiments, theheavy chain comprises the CDR-H1 having the amino acid sequence of SEQID NO: 1, CDR-H2 having the amino acid sequence of SEQ ID NO: 8 andCDR-H3 having the amino acid sequence of SEQ ID NO: 3. Hence, anysequence deviations to SEQ ID NO: 19 are located in the frameworkregions, but not in the CDRs. In particular, the heavy chain comprisesthe amino acid sequence of SEQ ID NO: 19.

In specific embodiments, the light chain comprises an amino acidsequence which is at least 90% identical to the amino acid sequence ofSEQ ID NO: 16. Especially, the light chain comprises an amino acidsequence which is at least 95%, in particular at least 98% identical tothe amino acid sequence of SEQ ID NO: 16. In these embodiments, thelight chain still comprises CDRs having the amino acid sequences of SEQID NOs: 4, 5 and 6. Hence, any sequence deviations to SEQ ID NO: 16 arelocated in the framework regions, but not in the CDRs. In particular,the light chain comprises the amino acid sequence of SEQ ID NO: 16.

In specific embodiments, the heavy chain has an amino acid sequencewhich is at least 90% identical to the amino acid sequence of SEQ ID NO:15, wherein the CDRs still have the amino acid sequences of SEQ ID NOs:1, 7 and 3, and the light chain variable region has an amino acidsequence which is at least 90% identical to the amino acid sequence ofSEQ ID NO: 16, wherein the CDRs still have the amino acid sequences ofSEQ ID NOs: 4, 5 and 6. In particular, the heavy chain has an amino acidsequence which is at least 95% identical to the amino acid sequence ofSEQ ID NO: 15, wherein the CDRs still have the amino acid sequences ofSEQ ID NOs: 1, 7 and 3, and the light chain has an amino acid sequencewhich is at least 95% identical to the amino acid sequence of SEQ ID NO:16, wherein the CDRs still have the amino acid sequences of SEQ ID NOs:4, 5 and 6.

In specific embodiments, the heavy chain has an amino acid sequencewhich is at least 90% identical to the amino acid sequence of SEQ ID NO:19, wherein the CDRs still have the amino acid sequences of SEQ ID NOs:1, 8 and 3, and the light chain has an amino acid sequence which is atleast 90% identical to the amino acid sequence of SEQ ID NO: 16, whereinthe CDRs still have the amino acid sequences of SEQ ID NOs: 4, 5 and 6.In particular, the heavy chain has an amino acid sequence which is atleast 95% identical to the amino acid sequence of SEQ ID NO: 19, whereinthe CDRs still have the amino acid sequences of SEQ ID NOs: 1, 8 and 3,and the light chain has an amino acid sequence which is at least 95%identical to the amino acid sequence of SEQ ID NO: 16, wherein the CDRsstill have the amino acid sequences of SEQ ID NOs: 4, 5 and 6.

The antibody includes and encompasses modified forms thereof. Themodified form of the antibody means an antibody provided with chemicalor biological modification. The chemically modified form includes a formhaving an amino acid skeleton conjugated with a chemical moiety, a formhaving a chemically modified N-linked or O-linked carbohydrate chain,and the like. Said chemical moiety or form can be toxic or cytotoxic.The biologically modified form includes a form that has undergonepost-translational modification (e.g., N-linked or O-linkedglycosylation, N-terminal or C-terminal processing, deamidation,isomerization of aspartic acid, or oxidation of methionine), a formcontaining a methionine residue added to the N-terminus by expressionusing prokaryotic host cells, and the like. Such a modified form is alsomeant to include a form labeled to permit detection or isolation of theantibody or the antigen, for example, an enzyme-labeled form, afluorescently labeled form, or an affinity-labeled form. Such a modifiedform of the antibody is useful in improvement in the stability or bloodretention of the original antibody, reduction in antigenicity, detectionor isolation of the antibody or the antigen, etc.

In particular, the antibody may comprise one or more modificationsselected from the group consisting of defucosylation, reduced fucose,N-linked glycosylation, O-linked glycosylation, N-terminal processing,C-terminal processing, deamidation, isomerization of aspartic acid,oxidation of methionine, substitutions of two leucine (L) residues toalanine (A) at position 234 and 235 (according to EU index) of the heavychain (LALA), amidation of a proline residue and deletion or lack ofone, two, or three amino acids at the carboxyl terminus. In specificembodiments, the antibody lacks one, two, or three carboxyl-terminalamino acid(s) at one or both heavy chains, or it lacks twocarboxyl-terminal amino acid and the carboxyl-terminal proline residuesis amidated at one or both heavy chains.

Such a modification may be made at an arbitrary position or the desiredposition in the antibody thereof. Alternatively, the same or two or moredifferent modifications may be made at one or two or more positionstherein.

For example, antibodies produced by cultured mammalian cells are knownto lack a carboxyl-terminal lysine residue in its heavy chain (Journalof Chromatography A, 705: 129-134 (1995)). It is also known thatoccasionally 2 carboxyl-terminal amino acid residues (i.e., glycine andlysine) of a heavy chain are missing and that a proline residue newlylocated at the carboxyl terminus is amidated (Analytical Biochemistry,360: 75-83 (2007)). Such lack or modification in these heavy chainsequences, however, affects neither the ability of the antibody to bindto its antigen nor the effector functions (complement activation,antibody-dependent cytotoxicity, etc.) of the antibody.

In certain embodiments, the antibody comprises a deletion or lack of 1or 2 amino acid(s) in the carboxyl terminus of the heavy chain, and hasan amidated residue (e.g., an amidated proline residue at thecarboxyl-terminal site of the heavy chain). However, the antibody is notlimited to the types described above as long as the deletion mutantmaintains the ability to bind to the antigen.

In certain embodiments, two heavy chains of the antibody may be composedof any one type of heavy chain selected from the group consisting of thefull length heavy chains and the heavy chains of the deletion mutant ormay be composed of the combination of any two types selected therefrom.The quantitative ratio of the deletion variant heavy chain(s) depends onthe type of cultured mammalian cells producing the antibody, and theculture conditions of the cells.

In specific embodiments, the antibody can include two heavy chains, bothof which lack one carboxyl-terminal amino acid residue.

In specific embodiments, the antibody comprises the heavy chain havingan amino acid sequence represented by amino acid Nos 1 to 446 of SEQ IDNO: 15 or 22 and the light chain having an amino acid sequencerepresented by amino acid Nos 1 to 219 of SEQ ID NO: 16. In certainembodiments, the amino acid at position 57 of SEQ ID NO: 15 is selectedfrom the group consisting of glutamine, alanine, valine, histidine,tryptophan, tyrosine, lysine and arginine; especially glutamine,histidine, tryptophan, tyrosine, lysine and arginine. Preferably, theamino acid at position 57 of SEQ ID NO: 15 is glutamine, histidine,tryptophan, lysine or arginine, especially glutamine.

In specific embodiments, the antibody comprises the heavy chain havingan amino acid sequence represented by amino acid Nos 1 to 446 of SEQ IDNO: 19 and the light chain having an amino acid sequence represented byamino acid Nos 1 to 219 of SEQ ID NO: 16.

In certain embodiments, the antibody competes for the binding to TA-MUC1with an antibody comprising a heavy chain variable region having theamino acid sequence of SEQ ID NO: 10 and a light chain variable regionhaving the amino acid sequence of SEQ ID NO: 12, or an antibodycomprising a heavy chain variable region having the amino acid sequenceof SEQ ID NO: 11 and a light chain variable region having the amino acidsequence of SEQ ID NO: 12.

In certain embodiments, the antibody has the following properties: (a)specifically binding to MUC1, and/or (b) having the activity of beinginternalized into MUC1-expressing cells through binding to MUC1. Incertain embodiments, the antibody comprises at least one antibody heavychain. Especially, the antibody comprises two antibody heavy chains. Theantibody heavy chains in particular comprise a VH domain, a CH1 domain,a hinge region, a CH2 domain and a CH3 domain. In certain otherembodiments, the antibody heavy chains comprise a CH2 domain and a CH3domain, but do not comprise a CH1 domain. In further embodiments, one ormore constant domains of the heavy chains may be replaced by otherdomains, in particular similar domains such as for example albumin. Theantibody heavy chains may be of any type, including γ-, α-, ε-, δ- andμ-chains, and preferably are γ-chains, including γ1-, γ2-, γ3- andγ4-chains, especially γ1-chains. Hence, the antibody preferably is anIgG-type antibody such as an IgG1-, IgG3- or IgG4-type antibody, inparticular an IgG1-type antibody.

In particular, the antibody further comprises at least one antibodylight chain, especially two antibody light chains. The antibody lightchains in particular comprise a VL domain and a CL domain. The antibodylight chain may be a κ-chain or a λ-chain and especially is a κ-chain.

In certain embodiments, the antibody comprises two antibody heavy chainsand two antibody light chains. In particular, the antibody comprises twoantibody heavy chains of the γ1-type, each comprising a VH domain, a CH1domain, a hinge region, a CH2 domain and a CH3 domain, and two antibodylight chains of the κ-type, each comprising a VL domain and a CL domain.

In alternative embodiments, the antibody does not comprise an antibodylight chain. In these embodiments, the light chain variable region maybe fused to the N terminus of the heavy chain variable region or isinserted C terminal to the heavy chain variable region. Peptide linkersmay be present to connect the light chain variable region with theremaining parts of the heavy chain.

In preferred embodiments, the antibody comprises an Fc region. Theantibody may especially be a whole antibody, comprising two heavy chainseach comprising the domains VH, CH1, hinge region, CH2 and CH3, and twolight chains each comprising the domains VL and CL. The antibody inparticular is capable of binding to one or more human Fcγ receptors,especially human Fcγ receptor IIIA. In alternative embodiments, theantibody does not or not significantly bind the human Fcγ receptor IIIA,and especially does not or not significantly bind to any human Fcγreceptor. In these embodiments the antibody in particular does notcomprise a glycosylation site in the CH2 domain.

In alternative embodiments, the antibody does not comprise an Fc region.In these embodiments, the antibody in particular is a single chainvariable region fragment (scFv) or another antibody fragment notcomprising an Fc region.

Glycosylation of the Anti-MUC1 Antibody

The anti-MUC1 antibody may comprise a CH2 domain in one or more antibodyheavy chains. Natural human antibodies of the IgG type comprise anN-glycosylation site in the CH2 domain. The CH2 domains present in theantibody may or may not comprise an N-glycosylation site. In certainembodiments, the antibody does not comprise a glycosylation site in theCH2 domain. In particular, the antibody does not comprise an asparagineresidue at the position in the heavy chain corresponding to position 297according to the IMGT/Eu numbering system. For example, the antibody maycomprise an Ala297 mutation in the heavy chain. In these embodiments,the antibody preferably has a strongly reduced ability or completelylacks the ability to induce, via binding to Fcγ receptors,antibody-dependent cellular cytotoxicity (ADCC) and/orantibody-dependent cellular phagocytosis (ADCP) and/orcomplement-dependent cytotoxicity (CDC). Strongly reduced ability inthis respect in particular refers to a reduction to 10% or less,especially 3% or less, 1% or less or 0.1% or less activity compared tothe same antibody comprising an N-glycosylation site in its CH2 domainsand having a common mammalian glycosylation pattern such as thoseobtainable by production in human cell lines or in CHO cell lines, forexample a glycosylation pattern as described herein. In theseembodiments, the antibody in particular is an IgG1-type antibody.

In alternative embodiments, the CH2 domains present in the antibodycomprise an N-glycosylation site. This glycosylation site in particularis at an amino acid position corresponding to amino acid position 297 ofthe heavy chain according to the IMGT/Eu numbering system and has theamino acid sequence motive Asn Xaa Ser/Thr wherein Xaa may be any aminoacid except proline. The N-linked glycosylation at Asn297 is conservedin mammalian IgGs as well as in homologous regions of other antibodyisotypes. Due to optional additional amino acids which may be present inthe variable region or other sequence modifications, the actual positionof this conserved glycosylation site may vary in the amino acid sequenceof the antibody. Preferably, the glycans attached to the antibody arebiantennary complex type N-linked carbohydrate structures, preferablycomprising at least the following structure:Asn-GlcNAc-GlcNAc-Man-(Man-GlcNAc)₂wherein Asn is the asparagine residue of the polypeptide portion of theantibody; GlcNAc is N-acetylglucosamine and Man is mannose. The terminalGlcNAc residues may further carry a galactose residue, which optionallymay carry a sialic acid residue. A further GlcNAc residue (namedbisecting GlcNAc) may be attached to the Man nearest to the polypeptide.A fucose may be bound to the GlcNAc attached to the Asn. In theseembodiments, the antibody in particular is an IgG1-type antibody.

In preferred embodiments, the antibody does not comprise N-glycolylneuraminic acids (NeuGc) or detectable amounts of NeuGc. Furthermore,the antibody preferably also does not comprise Galili epitopes(Galα1,3-Gal structures) or detectable amounts of the Galili epitope. Inparticular, the relative amount of glycans carrying NeuGc and/orGalα1,3-Gal structures is less than 0.1% or even less than 0.02% of thetotal amount of glycans attached to the CH2 domains of the antibodies inthe population of antibodies.

In particular, the antibody has a human glycosylation pattern. Due tothese glycosylation properties, foreign immunogenic non-human structureswhich induce side effects are absent which means that unwanted sideeffects or disadvantages known to be caused by certain foreign sugarstructures such as the immunogenic non-human sialic acids (NeuGc) or theGalili epitope (Gal-Gal structures), both known for rodent productionsystems, or other structures like immunogenic high-mannose structures asknown from e.g. yeast systems are avoided.

In specific embodiments, the antibody comprises a glycosylation patternhaving a detectable amount of glycans carrying a bisecting GlcNAcresidue. In particular, the relative amount of glycans carrying abisecting GlcNAc residue is at least 0.5%, especially at least 1% of thetotal amount of glycans attached to the glycosylation sites of theantibody in a composition. Furthermore, in certain embodiments theglycosylation pattern comprises a relative amount of glycans carrying atleast one galactose residue of at least 25% of the total amount ofglycans attached to the antibody in a composition. In particular, therelative amount of glycans carrying at least one galactose residue is atleast 30%, especially at least 35% or at least 40% of the total amountof glycans attached to the antibody in a composition. In specificembodiments, the glycosylation pattern comprises a relative amount ofglycans carrying at least one sialic acid residue of at least 1% of thetotal amount of glycans attached to the antibody in a composition. Inparticular, the relative amount of glycans carrying at least one sialicacid residue is at least 1.5%, especially at least 2% of the totalamount of glycans attached to the antibody in a composition.

The antibody may have a glycosylation pattern having a high amount ofcore fucose or a low amount of core fucose. A reduced amount offucosylation increases the ability of the antibody to induce ADCC. Incertain embodiments, the relative amount of glycans carrying a corefucose residue is 40% or less, especially 30% or less or 20% or less ofthe total amount of glycans attached to the antibody in a composition.In alternative embodiments, the relative amount of glycans carrying acore fucose residue is at least 60%, especially at least 65% or at least70% of the total amount of glycans attached to the antibody in acomposition.

Via the presence or absence of the glycosylation site in the CH2 domainof the anti-MUC1 antibody and the presence or absence of fucose in theglycan structures at said glycosylation site, the ability of theantibody to induce ADCC and the strength of said ADCC induction can becontrolled. The ADCC activity is increased by glycosylation of the Fcpart of the antibody and further by reducing the amount of fucosylationin said glycosylation. In certain applications, fine tuning of the ADCCactivity is important. Therefore, in certain situations, the antibodywithout a glycosylation site in the CH2 domain, the antibody with aglycosylation site in the CH2 domain and with a high amount offucosylation, or the antibody with a glycosylation site in the CH2domain and with a low amount of fucosylation may be most advantageous.

Production of the Anti-MUC1 Antibody

The antibody is preferably recombinantly produced in a host cell. Thehost cell used for the production of the antibody may be any host cellswhich can be used for antibody production. Suitable host cells are inparticular eukaryotic host cells, especially mammalian host cells.Exemplary host cells include yeast cells such as Pichia pastoris celllines, insect cells such as SF9 and SF21 cell lines, plant cells, birdcells such as EB66 duck cell lines, rodent cells such as CHO, NS0, SP2/0and YB2/0 cell lines, and human cells such as HEK293, PER.C6, CAP,CAP-T, AGE1.HN, Mutz-3 and KG1 cell lines.

In certain embodiments, the antibody is produced recombinantly in ahuman blood cell line, in particular in a human myeloid leukemia cellline. Preferred human cell lines which can be used for production of theantibody as well as suitable production procedures are described in WO2008/028686 A2. In a specific embodiment, the antibody is obtained byexpression in a human myeloid leukemia cell line selected from the groupconsisting of NM-H9D8, NM-H9D8-E6 and NM-H9D8-E6Q12 and cell linesderived therefrom. These cell lines were deposited under the accessionnumbers DSM ACC2806 (NM-H9D8; deposited on Sep. 15, 2006), DSM ACC2807(NM-H9D8-E6; deposited on Oct. 5, 2006) and DSM ACC2856 (NM-H9D8-E6Q12;deposited on Aug. 8, 2007) according to the requirements of the BudapestTreaty at the Deutsche Sammlung von Mikroorganismen and Zellkulturen(DSMZ), Inhoffenstraße 7B, 38124 Braunschweig (DE) by Glycotope GmbH,Robert-Rössle-Str. 10, 13125 Berlin (DE). NM-H9D8 cells provide aglycosylation pattern with a high degree of sialylation, a high degreeof bisecting GlycNAc, a high degree of galactosylation and a high degreeof fucosylation. NM-H9D8-E6 and NM-H9D8-E6Q12 cells provide aglycosylation pattern similar to that of NM-H9D8 cells, except that thedegree of fucosylation is very low. Other suitable cell lines includeK562, a human myeloid leukemia cell line present in the American TypeCulture Collection (ATCC CCL-243), as well as cell lines derived fromthe aforementioned.

In further embodiments, the antibody is produced recombinantly in CHOcells. Especially, the antibody may be produced recombinantly in a CHOdhfr− cell line such as the cell line of ATCC No. CRL-9096.

Conjugates of the Anti-MUC1 Antibody

According to the present invention, the antibody is conjugated to one ormore cytotoxic agents. The cytotoxic agent may be any cytotoxic agentsuitable for conjugation to the antibody. If more than one cytotoxicagent is present in the antibody, these cytotoxic agents may beidentical or different, and in particular are all identical. Conjugationof the cytotoxic agent to the antibody can be achieved using any methodsknown in the art. The cytotoxic agent may be covalently, in particularby fusion or chemical coupling, or non-covalently attached to theantibody. In certain embodiments, the cytotoxic agent is covalentlyattached to the antibody, especially via a linker moiety. The linkermoiety may be any chemical entity suitable for attaching the cytotoxicagent to the antibody.

In addition to the cytotoxic agent, the conjugate according to theinvention may further comprise a further agent conjugated thereto. Thefurther agent preferably is useful in therapy, diagnosis, prognosisand/or monitoring of a disease, in particular cancer. For example, thefurther agent may be selected from the group consisting ofradionuclides, chemotherapeutic agents, antibodies or antibodyfragments, in particular those of different specificity than theanti-MUC1 antibody, e.g. checkpoint antibodies which block or activateimmunomodulatory targets, enzymes, interaction domains, detectablelabels, toxins, cytolytic components, immunomodulators, immunoeffectors,MHC class I or class II antigens, and liposomes.

A particular preferred cytotoxic agent is a radionuclide or a cytotoxicagent capable of killing cancer cells, such as a chemotherapeutic agent.In certain preferred embodiments, a chemotherapeutic agent is attachedto the anti-MUC1 antibody forming a conjugate. Chemotherapeutic agent isnot particularly limited as long as the compound has an antitumor effectand has a substituent or a partial structure that can be connected to alinker structure. Upon cleavage of a part or the whole of the linker intumor cells, the chemotherapeutic agent or the antitumor compound moietyis released so that the chemotherapeutic agent exhibits an antitumoreffect. As the linker is cleaved at a connecting position with theagent, chemotherapeutic agent is released in its original structure toexert its original antitumor effect.

Specific examples of chemotherapeutic agents that can be conjugated ascytotoxic agent include alkylating agents such as cisplatin,anti-metabolites, plant alkaloids and terpenoids, vinca alkaloids,podophyllotoxin, taxanes such as taxol, topoisomerase inhibitors such asirinotecan and topotecan, antineoplastics such as doxorubicin ormicrotubule inhibitors such as maytansin/maytansinoids.

The chemotherapeutic agent may in particular be selected from a groupconsisting of a V-ATPase inhibitor, a pro-apoptotic agent, a Bcl2inhibitor, an MCL1 inhibitor, a HSP90 inhibitor, an IAP inhibitor, anmTor inhibitor, a microtubule stabilizer, a microtubule destabilizer, adolastatin, a maytansin, a maytansinoid, amatoxin, a methionineaminopeptidase, an inhibitor of nuclear export of proteins CRM1, a DPPIVinhibitor, proteasome inhibitors, inhibitors of phosphoryl transferreactions in mitochondria, a protein synthesis inhibitor, a kinaseinhibitor, a CDK2 inhibitor, a CDK9 inhibitor, a kinesin inhibitor, anHDAC inhibitor, a topoisomerase I inhibitor, a DNA damaging agent, a DNAalkylating agent, a DNA intercalator, a DNA minor groove binder, a DHFRinhibitor, an inhibitor of microtubule formation, a stabilizer ofmicrotubule, a stabilizer of actin, a topoisomerase II inhibitor, aplatinum compound, a ribosome inhibitor, an RNA polymerase II inhibitorand a bacterial toxin. In specific embodiments, the chemotherapeuticagent attached to the anti-MUC1 antibody is selected from the groupconsisting of a microtubule inhibitor such as maytansinoid, atopoisomerase I inhibitor, a DNA damaging agent, a DNA alkylating agentand a DNA minor groove binder.

In some embodiments of the chemotherapeutic agent is a maytansin ormaytansinoid. Specific examples of maytansinoids useful for conjugationinclude maytansinol,N^(2′)-deacetyl-N^(2′)-(3-mercapto-1-oxopropyl)-maytansine (DM1),N^(2′)-deacetyl-N^(2′)-(4-mercapto-1-oxopentyl)-maytansine (DM3), andN^(2′)-deacetyl-N^(2′)-(4-methyl-4-mercapto-1-oxopentyl)-maytansine(DM4). In particular, DM1 or DM4 is attached to the anti-MUC1 antibody.In some embodiments, the chemotherapeutic agent attached to theanti-MUC1 antibody is a DNA minor groove binder, in particularpyrrolobenzodiazepine (PBD), pyrrolobenzodiazepine dimer (PBD dimer),duocarmycin, duocarmycin-hydroxybenzamide-azaindole (DU BA),seco-duocarmycin-hydroxybenzamide-azaindole (seco-DUBA) or doxorubicin.In some embodiments, the chemotherapeutic agent attached to theanti-MUC1 antibody is a DNA alkylating agent, in particularindolinobenzodiazepine or oxazolidinobenzodiazepine. In someembodiments, the chemotherapeutic agent attached to the anti-MUC1antibody is a DNA damaging agent, in particular calicheamicin. In someembodiments, the chemotherapeutic agent attached to the anti-MUC1antibody is a topoisomerase I inhibitor, in particular camptothecin andits derivatives such as 7-ethyl-10-hydroxy-camptothecin (SN-38),(S)-9-dimethylaminomethyl-10-hydroxycamptothecin (topotecan),(1S,9S)-1-amino-9-ethyl-5-fluoro-1,2,3,9,12,15-hexahydro-9-hydroxy-4-methyl-10H,13H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinoline-10,13-dione(Exatecan (DX-8951)) andN-[(1S,9S)-9-Ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl]-2-hydroxyacetamide(DXd). In some embodiments, the chemotherapeutic agent attached to theanti-MUC1 antibody is an inhibitor of microtubule formation, inparticular a tubulysin, an ansamitocin, a podophyllotoxin or avinblastine. In some embodiments, the chemotherapeutic agent attached tothe anti-MUC1 antibody is a stabilizer of microtubule, in particular apaclitaxel or an epothilone. In some embodiments, the chemotherapeuticagent attached to the anti-MUC1 antibody is a stabilizer of actin, inparticular a phallotoxin. In some embodiments, the chemotherapeuticagent attached to the anti-MUC1 antibody is a topoisomerase 11inhibitor, in particular a teniposide, a XK469, a razoxane, anamsacrine, an idarubicin or a mebarone. In some embodiments, thechemotherapeutic agent attached to the anti-MUC1 antibody is a platinumcompound, in particular a cisplatin, a carboplatin, an oxaliplatin, anedaplatin, a triplatin tetranitrate, a phenanthriplatin, a picoplatinor a sattraplatin. In some embodiments, the chemotherapeutic agentattached to the anti-MUC1 antibody is a ribosome inhibitor, inparticular ricin, a saporin, an abrin, a diphtheria toxin or an exotoxinA. In some embodiments, the chemotherapeutic agent attached to theanti-MUC1 antibody is an RNA polymerase 11 inhibitor, in particular anamatoxin, such as, for example, an amanitin. In some embodiments, thechemotherapeutic agent attached to the anti-MUC1 antibody is a bacterialtoxin, in particular an anthrax toxin. Suitable antibody drug conjugatesare also described in EP 16 151 774.3 and LU 92659, to which isexplicitly referred to herewith.

In preferred embodiments, the chemotherapeutic agent is(1S,9S)-1-amino-9-ethyl-5-fluoro-1,2,3,9,12,15-hexahydro-9-hydroxy-4-methyl-10H,13H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinoline-10,13-dione (exatecan (DX-8951))or DXd.

Exatecan (DX-8951) is an antitumor compound represented by the followingformula:

The compounds can be easily obtained by, for example, a method describedin U.S. Patent Publication No. US2016/0297890 or other known methods,and the amino group at position 1 can be preferably used as a connectingposition to the linker structure. Further, Exatecan may be released intumor cells while a part of the linker is still attached thereto.However, the compound exerts an excellent antitumor effect even in sucha state.

DXd is a compound represented by the following formula:

Since exatecan or DXd has a camptothecin structure, it is known that theequilibrium shifts to a structure with a formed lactone ring (closedring) in an acidic aqueous medium (e.g., of the order of pH 3) whereasthe equilibrium shifts to a structure with an opened lactone ring (openring) in a basic aqueous medium (e.g., of the order of pH 10). A drugconjugate into which exatecan residues corresponding to such a closedring structure and an open ring structure have been introduced is alsoexpected to have an equivalent antitumor effect, and it is needless tosay that any of such drug conjugates is included in the scope of thepresent invention. In certain embodiments, the further agent is apolypeptide of protein. This polypeptide or protein may in particular befused to a polypeptide chain of the antibody. In certain embodiments,the further agent being a polypeptide or protein is fused to the Cterminus of an antibody light chain of the antibody. In embodimentswherein the antibody comprises two antibody light chains, a furtheragent being a polypeptide or protein may be fused to the C terminus ofeach of the two antibody light chains. In further embodiments, thefurther agent being a polypeptide or protein is fused to the C terminusof an antibody heavy chain of the antibody. In embodiments wherein theantibody comprises two antibody heavy chains, a further agent being apolypeptide or protein may be fused to the C terminus of each of the twoantibody heavy chains. The further agents may be identical or differentand in particular have the same amino acid sequence. Suitable examplesof such further agents being a polypeptide or protein may be selectedfrom the group consisting of cytokines, chemokines, antibodies, antigenbinding fragments, enzymes, and interaction domains.

In certain embodiments, the further agent being a polypeptide or proteinis a checkpoint antibody which blocks and/or triggers activatingsignals. Examples of respective targets include CD40, CD3, CD137(4-1BB), OX40, GITR, CD27, CD278 (ICOS), CD154 (CD40 ligand), CD270(HVEM) and CD258 (LIGHT) as activating targets, CTLA4, PD1, CD80, CD244,A2AR, B7-H3 (CD276), B7-H4 (VTCN1), BTLA, IDO, KIR, LAG3, TIM-3, VISTAand phosphatidylserine as inhibitory targets, and their respectiveligands such as PDL1. In specific examples, the anti-MUC1 antibodycomprises two heavy chains and two light chains as described herein,wherein a scFv fragment specifically binding to CD3 is fused to the Cterminus of each heavy chain; or wherein a scFv fragment specificallybinding to PDL1 is fused to the C terminus of each light chain.

In further embodiments, the further agent being a polypeptide or proteinis an immunomodulatory compound such as a chemokine, cytokine or growthfactor. Suitable cytokines in this respect include interferons such asinterferon-α, interferon-β and interferon-γ, and interleukins. Suitablegrowth factors include G-CSF and GM-CSF.

Specific examples of linkers includes the structures represented by anyof the following formulas (a) to (f):-(Succinimid-3-yl-N)—CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂—C(═O)—,  (a)-(Succinimid-3-yl-N)—CH₂CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂—C(═O)—,  (b)-(Succinimid-3-yl-N)—CH₂CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂—O—CH₂—C(═O)—,  (c)-(Succinimid-3-yl-N)—CH₂CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—O—CH₂—C(═O)—,  (d)-(Succinimid-3-yl-N)—CH₂CH₂—C(═O)—NH—CH₂CH₂O—CH₂CH₂O—CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂—C(═O)—,and  (e)-(Succinimid-3-yl-N)—CH₂CH₂—C(═O)—NH—CH₂CH₂O—CH₂CH₂O—CH₂CH₂O—CH₂CH₂O—CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂—C(═O)—,wherein -(Succinimid-3-yl-N)— has a structure represented by thefollowing formula:  (f)

In specific embodiments, linkers comprises the structures represented byany of the following formulas (a) to (c):-(Succinimid-3-yl-N)—CH₂CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂—O—CH₂—C(═O)—,  (a)-(Succinimid-3-yl-N)—CH₂CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—O—CH₂—C(═O)—,and  (b)-(Succinimid-3-yl-N)—CH₂CH₂—C(═O)—NH—CH₂CH₂O—CH₂CH₂O—CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂—C(═O)—.  (c)

In preferred embodiments, linkers comprises the structure represented byany of the following formula (a):-(Succinimid-3-yl-N)—CH₂CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂—O—CH₂—C(═O)—,  (a)

In alternative embodiment, the conjugate has a drug-linker structurerepresented by the following formula, wherein the antibody is conjugatedto a drug linker structure represented by the following formula by athioether bond, Asterisk* represents the point of connection to theantibody:

In preferred embodiment, the conjugate has a drug-linker structurerepresented by the following formula,

wherein AB represents the antibody, y represents an average number ofunits of the drug-linker structure conjugated to the antibody perantibody, the antibody is conjugated to a drug linker structurerepresented by the above formula by a thioether bond and the antibodyrepresents the aforementioned anti-MUC1 antibody, preferably theantibody being any one of the following combinations a) to d) of a heavychain variable region and a light chain variable region, or heavy chainand light chain:

-   -   (a) the heavy chain variable region has the amino acid sequence        of SEQ ID NO: 10 and the light chain variable region has the        amino acid sequence of SEQ ID NO: 12,    -   (b) the heavy chain variable region has the amino acid sequence        of SEQ ID NO: 11 and the light chain variable region has the        amino acid sequence of SEQ ID NO: 12,    -   (c) the heavy chain has the amino acid sequence of SEQ ID NO: 15        and the light chain has the amino acid sequence of SEQ ID NO:        16, and    -   (d) the heavy chain has the amino acid sequence of SEQ ID NO: 19        and the light chain has the amino acid sequence of SEQ ID NO:        16:

In the aforementioned conjugates, the number of conjugated drugmolecules (or cytotoxic agent) per antibody molecule is a key factorhaving an influence on the efficacy and safety thereof. The productionof the antibody-drug conjugate (or the conjugates) is carried out byspecifying reaction conditions such as the amounts of starting materialsand reagents used for reaction, so as to attain a constant number ofconjugated drug molecules. Unlike the chemical reaction of alow-molecular-weight compound, a mixture containing different numbers ofconjugated drug molecules is usually obtained. The number of conjugateddrug molecules per antibody molecule is defined and indicated as anaverage value, i.e., the average number of conjugated drug molecules.Unless otherwise specified, i.e., except in the case of representing anantibody-drug conjugate having a specific number of conjugated drugmolecules that is included in an antibody-drug conjugate mixture havingdifferent numbers of conjugated drug molecules, the number of conjugateddrug molecules according to the present invention also means an averagevalue as a rule. The number of exatecan molecules or DXd conjugated toan antibody molecule is controllable, and as an average number ofconjugated drug molecules per antibody, approximately 1 to 10 exatecanmolecules or 1 to 10 DXd can be conjugated. The number of exatecanmolecules or DXd is preferably 2 to 8, more preferably 4 to 8, furtherpreferably 7 to 8, and still further preferably 8. It is to be notedthat a person skilled in the art can design a reaction for conjugating arequired number of drug molecules to an antibody molecule based on thedescription of the examples of the present application, and can obtainan antibody-drug conjugate with a controlled number of conjugatedexatecan molecules.

In above preferred embodiment, after the conjugates are transferred tothe inside of tumor cells, the linker moiety is cleaved, then DXd isreleased to exert antitumor effect. (Clinical Cancer Research, 2016,Oct. 15; 22(20):5097-5108, Epub 2016 Mar. 29).

The conjugate labeled with various radioactive or non-radioactiveisotopes is also included in the present invention. One or more atomsconstituting the conjugate of the present invention may contain anatomic isotope at non-natural ratio. Examples of the atomic isotopeinclude deuterium (²H), tritium (³H), iodine-125 (¹²⁵I), and carbon-14(¹⁴C). Further, the conjugate may be radioactive-labeled with aradioactive isotope such as tritium (³H), iodine-125 (¹²⁵I), carbon-14(¹⁴C), copper 64 (⁶⁴Cu), zirconium-89 (⁸⁹Zr), iodine-124 (¹²⁴I),fluorine-18 (¹⁸F), indium-111 (¹¹¹I), carbon-11 (¹¹C) and iodine-131(¹³¹I). The conjugate labeled with a radioactive isotope is useful as atherapeutic or prophylactic agent, a reagent for research such as anassay reagent and an agent for diagnosis such as an in vivo diagnosticimaging agent. Without being related to radioactivity, any isotopevariant type of the conjugate is within the scope of the presentinvention.

The Nucleic Acid, Expression Cassette, Vector, Cell Line and Composition

The antibody part of the conjugate according to the invention may beencoded by a nucleic acid. The nucleic acid sequence of said nucleicacid may have any nucleotide sequence suitable for encoding theantibody. However, preferably the nucleic acid sequence is at leastpartially adapted to the specific codon usage of the host cell ororganism in which the nucleic acid is to be expressed, in particular thehuman codon usage. The nucleic acid may be double-stranded orsingle-stranded DNA or RNA, preferably double-stranded DNA such as cDNAor single-stranded RNA such as mRNA. It may be one consecutive nucleicacid molecule or it may be composed of several nucleic acid molecules,each coding for a different part of the antibody. The nucleotidesequence of heavy chain of PankoMab variant (PM-N54Q) may be representedby SEQ ID NO: 17 and nucleotide sequence of light chain of PankoMabvariant (PM-N54Q) may be represented by SEQ ID NO: 18.

If the antibody is composed of more than one different amino acid chain,such as a light chain and a heavy chain of the antibody, the nucleicacid may, for example, be a single nucleic acid molecule containingseveral coding regions each coding for one of the amino acid chains ofthe antibody, preferably separated by regulatory elements such as IRESelements in order to generate separate amino acid chains, or the nucleicacid may be composed of several nucleic acid molecules wherein eachnucleic acid molecule comprises one or more coding regions each codingfor one of the amino acid chains of the antibody. In addition to thecoding regions encoding the antibody, the nucleic acid may also comprisefurther nucleic acid sequences or other modifications which, forexample, may code for other proteins, may influence the transcriptionand/or translation of the coding region(s), may influence the stabilityor other physical or chemical properties of the nucleic acid, or mayhave no function at all.

An expression cassette or vector may comprise said nucleic acid and apromoter operatively connected with said nucleic acid. In addition, theexpression cassette or vector may comprise further elements, inparticular elements which are capable of influencing and/or regulatingthe transcription and/or translation of the nucleic acid, theamplification and/or reproduction of the expression cassette or vector,the integration of the expression cassette or vector into the genome ofa host cell, and/or the copy number of the expression cassette or vectorin a host cell. Suitable expression cassettes and vectors comprisingrespective expression cassettes for expressing antibodies are well knownin the prior art and thus, need no further description here.

A host cell may comprise the nucleic acid or the expression cassette orvector. The host cell may be any host cell. It may be an isolated cellor a cell comprised in a tissue. Preferably, the host cell is a culturedcell, in particular a primary cell or a cell of an established cellline, preferably a tumor-derived cell. Preferably, it is a bacterialcell such as E. coli, a yeast cell such as a Saccharomyces cell, inparticular S. cerevisiae, an insect cell such as a Sf9 cell, or amammalian cell, in particular a human cell such as a tumor-derived humancell, a hamster cell such as CHO, or a primate cell. In a preferredembodiment the host cell is derived from human myeloid leukaemia cells.Preferably, it is selected from the following cells or cell lines: K562,KG1, MUTZ-3 or a cell or cell line derived therefrom, or a mixture ofcells or cell lines comprising at least one of those aforementionedcells. The host cell is preferably selected from the group consisting ofNM-H9D8, NM-H9D8-E6, NM H9D8-E6Q12, and a cell or cell line derived fromanyone of said host cells. These cell lines and their properties aredescribed in detail in the PCT-application WO 2008/028686 A2. In furtherembodiments, the host cell is of a CHO dhfr-cell line such as the cellline of ATCC No. CRL-9096. In preferred embodiments, the host cell isoptimized for expression of glycoproteins, in particular antibodies,having a specific glycosylation pattern. Preferably, the codon usage inthe coding region of the nucleic acid and/or the promoter and thefurther elements of the expression cassette or vector are compatiblewith and, more preferably, optimized for the type of host cell used.Preferably, the antibody is produced by a host cell or cell line asdescribed above.

A method of producing the antibody may use a host cells as describedherein. The method in particular comprises the steps of providing a hostcell comprising a nucleic acid encoding the antibody, culturing the hostcell under conditions suitable for expression of the antibody, andobtaining the antibody expressed by the host cell. The antibodydescribed herein may be obtained or obtainable by said method.

In another aspect, the present invention provides a compositioncomprising the conjugate according to the invention. Furthermore, thecomposition may comprise one or more further components selected fromthe group consisting of solvents, diluents, and excipients Preferably,the composition is a pharmaceutical composition. In this embodiment, thecomponents of the composition preferably are all pharmaceuticallyacceptable. The composition may be a solid or fluid composition, inparticular a—preferably aqueous—solution, emulsion or suspension or alyophilized powder.

Use in Medicine

The conjugate in particular is useful in medicine, in particular intherapy, diagnosis, prognosis, detecting and/or monitoring of a disease,in particular a disease as described herein, preferably cancer,infections, inflammatory diseases, graft-versus-host disease andimmunodeficiencies.

Therefore, in a further aspect, the invention provides the conjugate orthe composition for use in medicine. Preferably, the use in medicine isa use in the treatment, prognosis, diagnosis, detecting and/ormonitoring of a disease such as, for example, diseases associated withabnormal cell growth such as cancer, infections such as bacterial,viral, fungal or parasitic infections, inflammatory diseases such asautoimmune diseases and inflammatory bowel diseases, and diseasesassociated with a reduce immune activity such as immunodeficiencies. Ina preferred embodiment, the disease is cancer.

Preferably, the cancer has a detectable expression of MUC1 (TA-MUC1),preferably detectable by immunohistochemistry, ELISA, RIA, enzyme-linkedimmunospot (ELISPOT) assay, dot blotting, Ouchterlony test orcounterimmunoelectrophoresis (CIE), or in-situ hybridization. Itespecially includes cells having an MUC1 (TA-MUC1) expression which isdetectable by immunohistochemistry or in-situ hybridization. The cancermay be tested on MUC1 (TA-MUC1) level prior to administration of theanti-MUC1 antibody.

The present invention further provides kits and devices comprising theconjugate according to the invention, and associated methods that areuseful in the diagnosis, detecting or monitoring of MUC1 associateddisorders such as cancer. In some embodiments, a sandwich ELISA kit fortesting or diagnosis comprising the conjugate of the present inventionis provided. This kit may further comprise one or more of a solution ofMUC1 (TA-MUC1) protein standards, a coloring reagent, a buffer solutionfor dilution, an antibody for solid phase, an antibody for detection,and a washing solution, and the like. Preferably, the amount of theconjugate bound to the antigen can be measured by the application of amethod such as an absorbance, fluorescence, luminescence, orradioisotope (RI) method. Preferably, an absorbance plate reader, afluorescence plate reader, a luminescence plate reader, an RI liquidscintillation counter, or the like is used in the measurement.

The antibody may be used for immunohistochemistry (IHC) analysis.

The immunohistochemistry is not particularly limited as long as thisapproach involves reacting a tissue section with an antigen-bindingantibody (primary antibody) and detecting the primary antibody boundwith the antigen.

Different forms of cancers including metastases can be treated with theconjugate according to the invention. The cancer can in particular beselected from the group consisting of colon cancer, lung cancer, ovariancancer, breast cancer such as triple negative breast cancer, pancreaticcancer, cervical cancer, endometrial cancer, gastrointestinal cancer,kidney cancer, head and neck cancer, thyroid cancer and urothelialcancer. The cancer may further in particular be selected from stomachcancer, liver cancer, bladder cancer, skin cancer, prostate cancer andblood cancer. In certain embodiments, the cancer is a metastasizingcancer. The cancer may include any type of metastases, such as skinmetastases, lymph node metastases, lung metastases, liver metastases,peritoneal metastases, pleural metastases and/or brain metastases. Incertain embodiments, the cancer has an inflammatory phenotype. In theseembodiments, any of the cancer types described above may be aninflammatory cancer.

In certain embodiments, the viral infection is caused by humanimmunodeficiency virus, herpes simplex virus, Epstein Barr virus,influenza virus, lymphocytic choriomeningitis virus, hepatitis B virusor hepatitis C virus. The inflammatory disease may be selected frominflammatory bowel disease, pelvic inflammatory disease, ischemicstroke, Alzheimer's disease, asthma, pemphigus vulgaris anddermatitis/eczema. The autoimmune disease may be selected from the groupconsisting of celiac disease, diabetes mellitus type 1, Graves' disease,inflammatory bowel disease, multiple sclerosis, psoriasis, rheumatoidarthritis, systemic lupus erythematosus, vitiligo, psoriatic arthritis,atopic dermatitis, scleroderma, sarcoidosis, primary biliary cirrhosis,Guillain-Barre syndrome, autoimmune hepatitis and ankylosingspondylitis. In certain embodiments, the disease comprises or isassociated with cells which express MUC1, especially TA-MUC1. Forexample, a cancer to be treated is MUC1 positive, especially TA-MUC1positive, i.e. comprises cancer cells which express MUC1, especiallyTA-MUC1.

In specific embodiments, the conjugate is used for treatment incombination with another therapeutic agent, especially for treatment ofcancer in combination with another anti-cancer agent. Said furthertherapeutic agent may be any known anti-cancer agent. Suitableanti-cancer therapeutic agents which may be combined with the conjugateaccording to the invention may be chemotherapeutic agents, otherantibodies, immunostimulatory agents, cytokines, chemokines, andvaccines. Furthermore, therapy with the conjugate may be combined withradiation therapy, surgery and/or traditional Chinese medicine.

Anti-cancer agents that can be used in combination with the conjugatemay be selected from any chemotherapeutic agent, in particularchemotherapeutic agents known to be effective for treatment of MUC1positive cancers. The type of chemotherapeutic agent also depends on thecancer to be treated. The combination partner may be selected from thegroup consisting of taxanes such as paclitaxel (Taxol), docetaxel(Taxotere) and SB-T-1214; cyclophosphamide; imatinib; pazopanib;capecitabine; cytarabine; vinorelbine; gemcitabine; anthracyclines suchas daunorubicin, doxorubicin, epirubicin, idarubicin, valrubicin andmitoxantrone; aromatase inhibitors such as aminoglutethimide,testolactone (Teslac), anastrozole (Arimidex), letrozole (Femara),exemestane (Aromasin), vorozole (Rivizor), formestane (Lentaron),fadrozole (Afema), 4-hydroxyandrostenedione,1,4,6-androstatrien-3,17-dione (ATD) and 4-androstene-3,6,17-trione(6-OXO); topoisomerase inhibitors such as irinotecan, topotecan,camptothecin, lamellarin D, etoposide (VP-16), teniposide, doxorubicin,daunorubicin, mitoxantrone, amsacrine, ellipticines, aurintricarboxylicacid and HU-331; platinum based chemotherapeutic agents such ascis-diamminedichloroplatinum(II) (cisplatin), cis-diammine(1,1-cyclobutanedicarboxylato)platinum(II) (carboplatin) and[(1R,2R)-cyclohexane-1,2-diamine](ethanedioato-O,O′)platinum(II)(oxaliplatin); PARP inhibitors such as olaparib, rucaparib andniraparib; TLR agonists such as imiquimod and resiquimod; andantimetabolites, in particular antifolates such as methotrexate,pemetrexed, raltitrexed and pralatrexate, pyrimidine analogues such asfluoruracil, gemcitabine, floxuridine, 5-fluorouracil andtegafur-uracil, and purine analogues, selective estrogen receptormodulators and estrogen receptor downregulators.

Furthermore, also therapeutic antibodies can be used as furthercombination partner. It may be any antibody that is useful in cancertherapy which is different from the anti-MUC1 antibody. In particular,the further antibody is approved for cancer treatment by anadministration such as the U.S. Food and Drug Administration (FDA), theEuropean Medicines Agency (EMA, formerly EMEA) and the Bundesinstitutfür Arzneimittel and Medizinprodukte (BfArM). Examples of the furtherantibody that can be used for combination treatment are anti-EGFRantibodies such as Cetuximab, Tomuzotuximab, Panitumumab, Zalutumumab,Nimotuzumab, Matuzumab and Necitumumab; anti-HER2 antibodies such asTrastuzumab, Timigutuzumab and Pertuzumab; anti-VEGF antibodies such asbevacizumab (Avastin); anti-CD52 antibodies such as alemtuzumab(Campath); anti-CD30 antibodies such as brentuximab (Adcetris);anti-CD33 antibodies such as gemtuzumab (Mylotarg); and anti-CD20antibodies such as rituximab (Rituxan, Mabthera), tositumomab (Bexxar)and ibritumomab (Zevalin). Further exemplary antibodies suitable forcombination with the cancer therapy described herein include antibodiesagainst antigens selected from the group consisting ofThomsen-Friedenreich antigen (TFα, TFβ), Tn, Lewis Y, CD44, folatereceptor α, NeuGc-GM3 ganglioside, DLL-3, RANKL, PTK7, Notch-3, EphrinA4, insulin-like growth factor receptor 1, activin receptor-likekinase-1, claudin-6, disialoganglioside GD2, endoglin, transmembraneglycoprotein NMB, CD56, tumor-associated calcium signal transducer 2,tissue factor, ectonucleotide pyrophosphatase/phosphodiesterase 3, CD70,P-cadherin, mesothelin, six transmembrane epithelial antigen of theprostate 1 (STEAP1), carcinoembryonic antigen-related cell adhesionmolecule 5 (CEACAM5), nectin 4, guanylyl cyclase C, solute carrierfamily 44 member 4 (SLC44A4), prostate-specific membrane antigen (PSMA),zinc transporter ZIP6 (LIV1 (ZIP6)), SLIT and NTRK-like protein 6(SLITRK6), trophoblast glycoprotein (TPBG; 5T4), Fyn3, carbonicanhydrase 9, NaPi2b, fibronectin extra-domain B, endothelin receptorETB, VEGFR2 (CD309), tenascin c, collagen IV and periostin.

The conjugate can further be combined with checkpoint antibodies, i.e.antibodies blocking or activating immunomodulatory targets. Thereby,inhibitory signals for an immune response can be blocked and/oractivating signals can be triggered. Examples of respective targetsinclude CD40, CD3, CD137 (4-1BB), OX40, GITR, CD27, CD278 (ICOS), CD154(CD40 ligand), CD270 (HVEM) and CD258 (LIGHT) as activating targets,CTLA4, PD1, CD80, CD244, A2AR, B7-H3 (CD276), B7-H4 (VTCN1), BTLA, IDO,KIR, LAG3, TIM-3, VISTA and phosphatidylserine as inhibitory targets,and their respective ligands such as PDL1.

In further embodiments, the conjugate can be combined with the treatmentwith immunomodulatory compounds such as chemokines, cytokines, growthfactors and vaccines. Suitable cytokines in this respect includeinterferons such as interferon-α, interferon-β and interferon-γ, andinterleukins. Suitable growth factors include G-CSF and GM-CSF.

The conjugate preferably is used for treatment of a primary tumor, arecurrent tumor and/or metastases of such tumors, and in particular isused for treatment before, during or after surgery and for theprevention or treatment of metastases. The conjugate in particular isfor the treatment of a patient as adjuvant therapy. In certainembodiments, the conjugate is for the treatment of a patient asneoadjuvant therapy or in a combined neoadjuvant-adjuvant therapy.Furthermore, the conjugate is for the treatment of a patient aspalliative therapy.

The cancer therapy with the conjugate preferably results in inhibitionof tumor growth and in particular reduction of tumor size. Furthermore,the occurrence of further metastases is prevented and/or their number isreduced by the treatment. The treatment preferably results in anincrease in progression-free survival; and/or an increase in lifespanand thus the overall survival.

The present invention further provides methods of therapy, diagnosis,prognosis, detecting and/or monitoring of a disease using the conjugateaccording to the invention. The embodiments and examples of the use ofthe conjugate in medicine also apply likewise to the medical methods. Inparticular, a method for treating a disease in a subject in need thereofcomprising administering to the subject a therapeutically effectiveamount of the conjugate according to the present invention is provided.

For example, the invention provides a method for treating cancer in asubject in need thereof comprising, administering to the subject withcancer a therapeutically effective amount of the conjugate according tothe invention. In specific embodiments, the cancer is characterized byexpressing TA-MUC1. The cancer may be selected from the group consistingof ovarian cancer, breast cancer, pancreatic cancer, lung cancer, coloncancer, stomach cancer, liver cancer, kidney cancer, blood cancer,endometrial cancer, thyroid cancer, leukemia, seminomas, melanomas,carcinomas, teratomas, lymphomas, sarcomas, mesotheliomas,neuroblastomas, gliomas, rectal cancer, adrenal cancer, skin cancer,cancer of the brain, cervical cancer, intestinal cancer, intestinecancer, head and neck cancer, gastrointestinal cancer, lymph nodecancer, esophagus cancer, colorectal cancer, ear, nose and throat (ENT)cancer, prostate cancer, bladder cancer, cancer of the uterus and themetastases thereof.

Furthermore, the invention provides a method for diagnosis, detecting ormonitoring cancer, comprising the step of contacting a test sample witha conjugate according to the invention.

Methods of Increasing the MUC1 Binding Affinity

A method of increasing the MUC1 binding affinity of an antibody maycomprise

-   -   (i) a heavy chain variable region comprising the        complementarity-determining regions (CDRs) CDR-H1 having the        amino acid sequence of SEQ ID NO: 1, CDR-H2 having the amino        acid sequence of SEQ ID NO: 8 and CDR-H3 having the amino acid        sequence of SEQ ID NO: 3, and    -   (ii) a light chain variable region comprising the        complementarity-determining regions (CDRs) CDR-L1 having the        amino acid sequence of SEQ ID NO: 4, CDR-L2 having the amino        acid sequence of SEQ ID NO: 5 and CDR-L3 having the amino acid        sequence of SEQ ID NO: 6,

the method comprising the step of substituting the amino acid residue atposition 8 of CDR-H2 with any amino acid residue except asparagine,resulting in CDR-H2 having the amino acid sequence of SEQ ID NO: 2.

The antibody which MUC1 binding affinity is to be increased inparticular is an antibody capable of binding to MUC1 as describedherein, except that it comprises an asparagine at position 8 of theCDR-H2 sequence.

In certain embodiments, the heavy chain variable region of the antibodywhich MUC1 binding affinity is to be increased comprises an amino acidsequence which is at least 90% identical to the amino acid sequence ofSEQ ID NO: 11. Especially, the heavy chain variable region comprises anamino acid sequence which is at least 95%, in particular at least 98%identical to the amino acid sequence of SEQ ID NO: 11. In theseembodiments, the heavy chain variable region still comprises CDRs havingthe amino acid sequences of SEQ ID NOs: 1, 8 and 3. Hence, any sequencedeviations to SEQ ID NO: 11 are located in the framework regions, butnot in the CDRs. In particular, the heavy chain variable regioncomprises the amino acid sequence of SEQ ID NO: 11.

In certain embodiments, the light chain variable region of the antibodywhich MUC1 binding affinity is to be increased comprises an amino acidsequence which is at least 90% identical to the amino acid sequence ofSEQ ID NO: 12. Especially, the light chain variable region comprises anamino acid sequence which is at least 95%, in particular at least 98%identical to the amino acid sequence of SEQ ID NO: 12. In theseembodiments, the light chain variable region still comprises CDRs havingthe amino acid sequences of SEQ ID NOs: 4, 5 and 6. Hence, any sequencedeviations to SEQ ID NO: 12 are located in the framework regions, butnot in the CDRs. In particular, the light chain variable regioncomprises the amino acid sequence of SEQ ID NO: 12.

In specific embodiments, the heavy chain variable region of the antibodywhich MUC1 binding affinity is to be increased has an amino acidsequence which is at least 90% identical to the amino acid sequence ofSEQ ID NO: 11, wherein the CDRs still have the amino acid sequences ofSEQ ID NOs: 1, 8 and 3, and the light chain variable region has an aminoacid sequence which is at least 90% identical to the amino acid sequenceof SEQ ID NO: 12, wherein the CDRs still have the amino acid sequencesof SEQ ID NOs: 4, 5 and 6. In particular, the heavy chain variableregion has an amino acid sequence which is at least 95% identical to theamino acid sequence of SEQ ID NO: 11, wherein the CDRs still have theamino acid sequences of SEQ ID NOs: 1, 8 and 3, and the light chainvariable region has an amino acid sequence which is at least 95%identical to the amino acid sequence of SEQ ID NO: 12, wherein the CDRsstill have the amino acid sequences of SEQ ID NOs: 4, 5 and 6.

For example, the antibody which MUC1 binding affinity is to be increasedis an anti-MUC1 antibody as disclosed in WO 2004/065423 A2 or WO2011/012309 A1. In particular, the antibody which MUC1 binding affinityis to be increased is gatipotuzumab or PankoMab.

The antibody which MUC1 binding affinity is increased in particular isan antibody capable of binding to MUC1 as described herein.

In certain embodiments, MUC1 binding is as described herein. Increasingthe MUC1 binding affinity in particular refers to an increase of atleast 10%, at least 20%, at least 33% or at least 50%. In preferredembodiments, MUC1 binding affinity is increased by at least 50%. TheMUC1 binding affinity may be determined as described in the examples,especially using surface plasmon resonance analysis or switchSENSE®technology (DRX2 Biosensor, manufactured by Dynamic Biosensors GmbH), asdescribed, e.g., in example 4a and b.

In certain embodiments, the step of substituting the amino acid residueat position 8 of CDR-H2 is achieved by introducing a mutation into thenucleic acid coding for the antibody, wherein the mutation is introducedin the codon coding for said amino acid residue. Introducing themutation can be done by any method. Several suitable methods are knownin the art and the skilled person is capable of performing the necessarytasks to introduce the mutation. The antibody with increased MUC1binding affinity can then be obtained by expressing the mutated nucleicacid, for example in a host cell. Nucleic acids, host cells and methodsfor producing the antibody are described herein and can be used for themethod for increasing the MUC1 binding affinity.

In specific embodiments, the method of increasing the MUC1 bindingaffinity of an antibody comprises the steps of

-   -   (a) providing a nucleic acid coding for the antibody which MUC1        binding affinity is to be increased    -   (b) introducing a mutation into said nucleic acid to produce a        mutated nucleic acid, wherein the mutation is introduced in the        codon coding for the amino acid residue at position 8 of CDR-H2        so that said codon codes for any amino acid residue except        asparagine; and    -   (c) expressing the mutated nucleic acid to produce an antibody        with increased MUC1 binding affinity.

A method of producing an antibody with increased MUC1 binding affinitymay comprise

-   -   (a) providing a nucleic acid coding for an antibody which        comprises        -   (i) a heavy chain variable region comprising the            complementarity-determining regions (CDRs) CDR-H1 having the            amino acid sequence of SEQ ID NO: 1, CDR-H2 having the amino            acid sequence of SEQ ID NO: 8 and CDR-H3 having the amino            acid sequence of SEQ ID NO: 3, and        -   (ii) a light chain variable region comprising the            complementarity-determining regions (CDRs) CDR-L1 having the            amino acid sequence of SEQ ID NO: 4, CDR-L2 having the amino            acid sequence of SEQ ID NO: 5 and CDR-L3 having the amino            acid sequence of SEQ ID NO: 6;    -   (b) introducing a mutation into said nucleic acid to produce a        mutated nucleic acid, wherein the mutation is introduced in the        codon coding for the amino acid residue at position 8 of CDR-H2        so that said codon codes for any amino acid residue except        asparagine; and    -   (c) producing the antibody with increased MUC1 binding affinity        by expressing the mutated nucleic acid in a host cell.

The embodiments, features and examples described herein for the otheraspects, especially for the method of increasing the MUC1 bindingaffinity of an antibody, also likewise apply to the method of producingan antibody with increased MUC1 binding affinity.

In certain embodiments, the method of producing an antibody withincreased MUC1 binding affinity further comprises a step (d) ofprocessing the antibody with increased MUC1 binding affinity.

For example, processing the antibody with increased MUC1 bindingaffinity may include isolating the antibody from the cell culture.Isolation of the antibody in particular refers to the separation of theantibody from the remaining components of the cell culture. Separationof the antibody from the cell culture medium may be performed, forexample, by chromatographic methods. Suitable methods and means forisolating antibodies are known in the art and can be readily applied bythe skilled person.

The obtained antibody may optionally be subject to further processingsteps such as e.g. modification steps such as chemical or enzymaticcoupling of a further agent to the antibody, and/or formulation steps inorder to produce the antibody in the desired quality and composition.Such further processing steps and methods are generally known in theart.

In further embodiments, step (d) additionally comprises the step ofproviding a pharmaceutical formulation comprising the antibody.Providing a pharmaceutical formulation comprising the antibody orformulating the antibody as a pharmaceutical composition in particularcomprises exchanging the buffer solution or buffer solution componentsof the composition comprising the antibody. Furthermore, this step mayinclude lyophilization of the antibody. In particular, the antibody istransferred into a composition only comprising pharmaceuticallyacceptable ingredients.

Production Method 1

The antibody-drug conjugate represented by the formula (1) given belowin which the antibody is connected to the linker structure via athioether can be produced by reacting an antibody having a sulfhydrylgroup converted from a disulfide bond by the reduction of the antibody,with the compound (2) obtainable by a known method (e.g., obtainable bya method described in the patent publication literature US2016/297890(e.g., a method described in paragraphs [0336] to [0374])). Thisantibody-drug conjugate can be produced by the following method, forexample.

wherein AB represents an antibody with a sulfhydryl group (3a), wherein

L1 has a structure represented by -(Succinimid-3-yl-N)—, and

L1′ represents a maleimidyl group represented by the following formula.

-   -   L1-LX has a structure represented by any of the following        formulas:        -(Succinimid-3-yl-N)—CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂—C(═O)—,        -(Succinimid-3-yl-N)—CH₂CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂—C(═O)—,        -(Succinimid-3-yl-N)—CH₂CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂—O—CH₂—C(═O)—,        -(Succinimid-3-yl-N)—CH₂CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—O—CH₂—C(═O)—,        -(Succinimid-3-yl-N)—CH₂CH₂—C(═O)—NH—CH₂CH₂O—CH₂CH₂O—CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂—C(═O)—,        and        -(Succinimid-3-yl-N)—CH₂CH₂—C(═O)—NH—CH₂CH₂O—CH₂CH₂O—CH₂CH₂O—CH₂CH₂O—CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂—C(═O)—

Among them, more preferred are the following:-(Succinimid-3-yl-N)—CH₂CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂—O—CH₂—C(═O)—,-(Succinimid-3-yl-N)—CH₂CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—O—CH₂—C(═O)—,and-(Succinimid-3-yl-N)—CH₂CH₂—C(═O)—NH—CH₂CH₂O—CH₂CH₂O—CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂—C(═O)—.

Further preferred are the following:-(Succinimid-3-yl-N)—CH₂CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂—O—CH₂—C(═O)—, and-(Succinimid-3-yl-N)—CH₂CH₂—C(═O)—NH—CH₂CH₂O—CH₂CH₂O—CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂—C(═O)—.

(NH-DX) has a structure represented by the following formula:

and it represents a group that is derived by removing one hydrogen atomof the amino group at position 1 of exatecan. In the above-describedreaction scheme (formula 8), the compound of formula (1) can beinterpreted as having a structure in which one structure moiety from thedrug to the linker terminus is connected to one antibody. However, thisdescription is given for the sake of convenience, and there are actuallymany cases in which a plurality of the aforementioned structure moietiesis connected to one antibody molecule. The same holds true for theexplanation of the production method described below.

Specifically, the antibody-drug conjugate (1) can be produced byreacting the compound (2) obtainable by a known method (e.g., obtainableby a method described in the patent publication literature US2016/297890(e.g., a method described in paragraphs [0336] to [0374])), with theantibody (3a) having a sulfhydryl group.

Provision of the sulfhydryl group on the antibody (3a) can beaccomplished by a method well known to a person skilled in the art(Hermanson, G. T, Bioconjugate Techniques, pp. 56-136, pp. 456-493,Academic Press (1996)). Examples of the method can include, but are notlimited to: Traut's reagent is reacted with the amino group of theantibody; N-succinimidyl S-acetylthioalkanoates are reacted with theamino group of the antibody followed by reaction with hydroxylamine;N-succinimidyl 3-(pyridyldithio)propionate is reacted with the antibody,followed by reaction with a reducing agent; the antibody is reacted witha reducing agent such as dithiothreitol, 2-mercaptoethanol, ortris(2-carboxyethyl)phosphine hydrochloride (TCEP) to reduce theinterchain disulfide bond in the antibody, so as to form a sulfhydrylgroup.

Specifically, an antibody with interchain disulfide bonds partially orcompletely reduced can be obtained by using 0.3 to 3 molar equivalentsof TCEP as a reducing agent per interchain disulfide bond in theantibody, and reacting the reducing agent with the antibody in a buffersolution containing a chelating agent. Examples of the chelating agentcan include ethylenediaminetetraacetic acid (EDTA) anddiethylenetriaminepentaacetic acid (DTPA). The chelating agent can beused at a concentration of 1 mM to 20 mM. A solution of sodiumphosphate, sodium borate, sodium acetate, or the like can be used as thebuffer solution. As a specific example, the antibody (3a) havingpartially or completely reduced sulfhydryl groups can be obtained byreacting the antibody with TCEP at 4° C. to 37° C. for 1 to 4 hours.

It is to be noted that by carrying out an addition reaction of asulfhydryl group to a drug-linker moiety, the drug-linker moiety can beconjugated by a thioether bond.

Then, using 2 to 20 molar equivalents of the compound (2) per antibody(3a) having a sulfhydryl group, the antibody-drug conjugate (1) in which2 to 8 drug molecules are conjugated per antibody can be produced.Specifically, a solution containing the compound (2) dissolved thereinmay be added to a buffer solution containing the antibody (3a) having asulfhydryl group for the reaction. In this context, a sodium acetatesolution, sodium phosphate, sodium borate, or the like can be used asthe buffer solution. pH for the reaction is 5 to 9, and more preferably,the reaction may be performed near pH 7. An organic solvent such asdimethyl sulfoxide (DMSO), dimethylformamide (DMF), dimethylacetamide(DMA), or N-methyl-2-pyrrolidone (NMP) can be used as a solvent fordissolving the compound (2). The reaction may be performed by adding thesolution containing the compound (2) dissolved in the organic solvent at1 to 20% v/v to a buffer solution containing the antibody (3a) having asulfhydryl group. The reaction temperature is 0 to 37° C., morepreferably 10 to 25° C., and the reaction time is 0.5 to 2 hours. Thereaction can be terminated by deactivating the reactivity of unreactedcompound (2) with a thiol-containing reagent. The thiol-containingreagent is, for example, cysteine or N-acetyl-L-cysteine (NAC). Morespecifically, the reaction can be terminated by adding 1 to 2 molarequivalents of NAC to the compound (2) used, and incubating the obtainedmixture at room temperature for 10 to 30 minutes.

Identification of Antibody-Drug Conjugate

The produced antibody-drug conjugate (e.g., antibody-drug conjugate (1))can be subjected to concentration, buffer exchange, purification, andmeasurement of antibody concentration and an average number ofconjugated drug molecules per antibody molecule according to commonprocedures described below, to identify the antibody-drug conjugate (1).

1. Common Procedure A: Concentration of Aqueous Solution of Antibody orAntibody-Drug Conjugate.

To Amicon Ultra (50,000 MWCO, Millipore Corporation) container, asolution of an antibody or an antibody-drug conjugate was added, and thesolution of the antibody or the antibody-drug conjugate was concentratedby centrifugation (centrifugation at 2000 G to 4000 G for 5 to 30minutes) using a centrifuge (Allegra X-15R, Beckman Coulter, Inc.).

2. Common Procedure B: Measurement of Antibody Concentration

Using a UV detector (Nanodrop 1000, Thermo Fisher Scientific Inc.),measurement of the antibody concentration was carried out according tothe method defined by the manufacturer. In this respect, 280 nmabsorption coefficient differing among antibodies (1.3 mLmg-1 cm-1 to1.8 mLmg-1 cm-1) was used.

3. Common Procedure C: Buffer Exchange for Antibody

A NAP-25 column (Cat. No. 17-0852-02, GE Healthcare Japan Corporation)using Sephadex G-25 carrier was equilibrated with a phosphate buffer (50mM, pH 6.0) (referred to as PBS6.0/EDTA in the present description)containing sodium chloride (50 mM) and EDTA (2 mM) according to themethod defined by the manufacturer. An aqueous solution of the antibodywas applied in an amount of 2.5 mL per NAP-25 column, and thereafter, afraction (3.5 mL) eluted with 3.5 mL of PBS6.0/EDTA was collected. Thisfraction was concentrated by common procedure A. After measurement ofthe concentration of the antibody using common procedure B, the antibodyconcentration was adjusted to 20 mg/mL using PBS6.0/EDTA.

4. Common Procedure D: Purification of Antibody-Drug Conjugate

A NAP-25 column was equilibrated with any commercially available buffersolution such as an acetate buffer containing sorbitol (5%) (10 mM, pH5.5; referred to as ABS in the present description). An aqueous reactionsolution of the antibody-drug conjugate (approximately 2.5 mL) wasapplied to the NAP-25 column, and thereafter, elution was carried outwith the buffer solution in an amount defined by the manufacturer, so asto collect an antibody fraction. A gel filtration purification process,in which the collected fraction was applied again to the NAP-25 column,and elution was carried out with the buffer solution, was repeated atotal of 2 or 3 times to obtain the antibody-drug conjugate excludingnon-conjugated drug linker and low-molecular-weight compounds(tris(2-carboxyethyl) phosphine hydrochloride (TCEP),N-acetyl-L-cysteine (NAC), and dimethyl sulfoxide).

5. Common Procedure E: Measurement of Antibody Concentration inAntibody-Drug Conjugate and Average Number of Conjugated Drug MoleculesPer Antibody Molecule

The conjugated drug concentration in the antibody-drug conjugate can becalculated by measuring UV absorbance of an aqueous solution of theantibody-drug conjugate at two wavelengths of 280 nm and 370 nm, andthereafter performing a calculation shown below.

The total absorbance at any given wavelength is equal to the sum of theabsorbance of all light-absorbing chemical species that are present in asystem [additivity of absorbance]. Therefore, based on the hypothesisthat the molar absorption coefficients of the antibody and the drug donot vary between before and after conjugation between the antibody andthe drug, the antibody concentration and the drug concentration in theantibody-drug conjugate are represented by the following equations:A ₂₈₀ =A _(D,280) +A _(A,280)=ε_(D,280) C _(D)+ε_(A,280) C_(A)  Equation (1)A ₃₇₀ =A _(D,370) +A _(A,370)=ε_(D,370) C _(D)+ε_(A,370) C_(A)  Equation (2)

In this context, A280 represents the absorbance of an aqueous solutionof the antibody-drug conjugate at 280 nm, A370 represents the absorbanceof an aqueous solution of the antibody-drug conjugate at 370 nm, AA, 280represents the absorbance of the antibody at 280 nm, A_(A,370)represents the absorbance of the antibody at 370 nm, A_(D,280)represents the absorbance of a conjugate precursor at 280 nm, A_(D,370)represents the absorbance of the conjugate precursor at 370 nm,ε_(A,280) represents the molar absorption coefficient of the antibody at280 nm, ε_(A,370) represents the molar absorption coefficient of theantibody at 370 nm, ε_(D,280) represents the molar absorptioncoefficient of the conjugate precursor at 280 nm, ε_(D,370) representsthe molar absorption coefficient of the conjugate precursor at 370 nm,C_(A) represents the antibody concentration in the antibody-drugconjugate, and C_(D) represents the drug concentration in theantibody-drug conjugate.

In this context, with regard to ε_(A,280), ε_(A,370), ε_(D,280), andε_(D,370), preliminarily prepared values (estimated values based oncalculation or measurement values obtained by UV measurement of thecompound) are used. For example, ε_(A,280) can be estimated from theamino acid sequence of the antibody by a known calculation method(Protein Science, 1995, vol. 4, 2411-2423). ε_(A,370) is generally zero.ε_(D,280) and ε_(D,370) can be obtained according to Lambert-Beer's law(Absorbance=Molar concentration×Molar absorption coefficient×Cell pathlength) by measuring the absorbance of a solution in which the conjugateprecursor used is dissolved at a certain molar concentration. C_(A) andC_(D) can be determined by measuring A₂₈₀ and A₃₇₀ of an aqueoussolution of the antibody-drug conjugate, and then solving thesimultaneous equations (1) and (2) by substitution of these values.Further, by diving C_(D) by C_(A), the average number of conjugated drugmolecules per antibody can be determined.

6. Common Procedure F: Measurement of Average Number of Conjugated DrugMolecules Per Antibody Molecule in Antibody-Drug Conjugate—(2)

The average number of conjugated drug molecules per antibody molecule inthe antibody-drug conjugate can also be determined by high-performanceliquid chromatography (HPLC) analysis using the following method, inaddition to the aforementioned “5. Common procedure E”. Hereinafter, themethod for measuring the average number of conjugated drug molecules byHPLC when the antibody is conjugated to the drug linker by a disulfidebond will be described. A person skilled in the art is capable ofappropriately measuring the average number of conjugated drug moleculesby HPLC, depending on the connecting pattern between the antibody andthe drug linker, with reference to this method.

F-1. Preparation of Sample for HPLC Analysis (Reduction of Antibody-DrugConjugate)

An antibody-drug conjugate solution (approximately 1 mg/mL, 60 μL) ismixed with an aqueous solution of dithiothreitol (DTT) (100 mM, 15 μL).By incubating the mixture at 37° C. for 30 minutes, the disulfide bondbetween the light chain and heavy chain of the antibody-drug conjugateis cleaved. The resulting sample is used in HPLC analysis.

F-2. HPLC Analysis

The HPLC analysis is carried out under the following measurementconditions.

HPLC system: Agilent 1290 HPLC system (Agilent Technologies, Inc.)

Detector: Ultraviolet absorption spectrometer (measurement wavelength:280 nm)

Column: ACQUITY UPLC BEH Phenyl (2.1×50 mm, 1.7 μm, 130 angstroms;Waters Corp., P/N 186002884)

Column temperature: 80° C.

Mobile phase A: Aqueous solution containing 0.10% trifluoroacetic acid(TFA) and 15% 2-propanol

Mobile phase B: Acetonitrile solution containing 0.075% TFA and 15%2-propanol Gradient program: 14%-36% (0 min-15 min), 36%-80% (15 min-17min), 80%-14% (17 min-17.01 min.), and 14% (17.01 min-25 min)

Sample injection: 10 μL

Or

HPLC system: Agilent 1290 HPLC system (Agilent Technologies, Inc.)

Detector: Ultraviolet absorption spectrometer (measurement wavelength:280 nm)

Column: PLRP-S (2.1×50 mm, 8 μm, 1000 angstroms; Agilent Technologies,Inc., P/N PL1912-1802)

Column temperature: 80° C.

Mobile phase A: 0.04% aqueous TFA solution

Mobile phase B: Acetonitrile solution containing 0.04% TFA

Gradient program: 29%-36% (0 min-12.5 min), 36%-42% (12.5 min-15 min),42%-29% (15 min-15.1 min), and 29%-29% (15.1 min-25 min)

Sample injection: 15 μL

F-3. Data Analysis

F-3-1. The light chain and heavy chain of the antibody are representedby Li and Hi, respectively, according to the number of conjugated drugmolecules (wherein i represents the number of conjugated drug molecules,i.e., the number of conjugated drug molecules according to the presentinvention is represented by L0, L1, H0, H1, H2, H3, etc.).

Compared with non-conjugated antibody light (L₀) and heavy (H₀) chains,a light chain bound to one drug molecule (L₁), a heavy chain bound toone drug molecule (H₁), a heavy chain bound to two drug molecules (H₂),and a heavy chain bound to three drug molecules (H₃) exhibit higherhydrophobicity in proportion to the number of conjugated drug moleculesand thus have a larger retention time. These chains are therefore elutedin the order of L₀ and L₁ or H₀, H₁, H₂, and H₃. Detection peaks can beassigned to any of L₀, L₁, H₀, H₁, H₂, and H₃ by the comparison ofretention times with L₀ and H₀.

F-3-2. Since the drug linker absorbs UV, peak area values are correctedin response to the number of conjugated drug linker molecules accordingto the following expression using the molar absorption coefficients ofthe light chain or heavy chain and the drug linker.

$\begin{matrix}{{{Corrected}{vale}{of}{peak}{area}{of}}{{light}{chain}{bond}{to}i{drug}{{molecule}(s)}({ALi})}} & \lbrack {{Expression}1} \rbrack\end{matrix}$$A_{Li} = {{Peak}{area} \times \frac{\varepsilon_{L,280}}{\varepsilon_{L,280} + {\varepsilon_{D,280} \times i}}}$

ε_(L,280): Molar absorption coefficient of light chain at 280 nm

ε_(D,280): Molar absorption coefficient of drug linker at 280 nm

i: The number of conjugated drug molecule(s)

$\begin{matrix}{{{Corrected}{vale}{of}{peak}{area}{of}}{{light}{chain}{bond}{to}i{drug}{{molecule}(s)}( A_{Hi} )}} & \lbrack {{Expression}2} \rbrack\end{matrix}$$A_{Hi} = {{Peak}{area} \times \frac{\varepsilon_{H,280}}{\varepsilon_{H,280} + {\varepsilon_{D,280} \times i}}}$

ε_(H,280): Molar absorption coefficient of heavy chain at 280 nm

ε_(D,280): Molar absorption coefficient of drug linker at 280 nm

i: The number of conjugated drug molecule(s)

In this context, a value estimated from the amino acid sequence of thelight chain or heavy chain of each antibody by a known calculationmethod (Protein Science, 1995, vol. 4, 2411-2423) can be used as themolar absorption coefficient (280 nm) of the light chain or heavy chainof the antibody. The actually measured molar absorption coefficient (280nm) of a compound in which the maleimide group has been converted tosuccinimide thioether by the reaction of each drug linker withmercaptoethanol or N-acetylcysteine was used as the molar absorptioncoefficient (280 nm) of the drug linker. The wavelength for absorbancemeasurement can be appropriately set by a person skilled in the art, butis preferably a wavelength at which the peak of the antibody can bemeasured, and more preferably 280 nm.

F-3-3. The peak area ratio (%) of each chain is calculated for the totalof the corrected values of peak areas according to the followingexpression.

$\begin{matrix}{{{Peak}{area}{ratio}{of}{light}{chain}{bond}{to}i{{drug}(s)}} = {\frac{A_{Li}}{A_{L0} + A_{L1}} \times 100}} & \lbrack {{Expression}3} \rbrack\end{matrix}$${{Peak}{area}{ratio}{of}{heavy}{chain}{bond}{to}i{{drug}(s)}} = {\frac{A_{Hi}}{A_{H0} + A_{H1} + A_{H2} + A_{H3}} \times 100}$

F-3-4. The average number of conjugated drug molecules per antibodymolecule in the antibody-drug conjugate is calculated according to thefollowing expression.Average number of conjugated drug molecules=(L ₀ peak area ratio×0+L ₀peak area ratio×1+H ₀ peak area ratio×0+H ₁ peak area ratio×1+H ₂ peakarea ratio×2+H ₃ peak area ratio×3)/100×2

It is to be noted that, in order to secure the amount of the conjugate,a plurality of conjugates having almost the same average number ofconjugated drug molecules (e.g., on the order of ±1), which have beenproduced under similar conditions, can be mixed to prepare a new lot. Inthis case, the average number of drug molecules falls between theaverage numbers of drug molecules before mixing.

SPECIFIC EMBODIMENTS

In the following, specific embodiments of antibody part of the conjugateaccording to the present invention are described.

Embodiment 1. An antibody capable of binding to MUC1, which comprises

-   -   (i) a heavy chain variable region comprising the        complementarity-determining regions (CDRs) CDR-H1 having the        amino acid sequence of SEQ ID NO: 1, CDR-H₂ having the amino        acid sequence of SEQ ID NO: 2 and CDR-H₃ having the amino acid        sequence of SEQ ID NO: 3, and    -   (ii) a light chain variable region comprising the        complementarity-determining regions (CDRs) CDR-L1 having the        amino acid sequence of SEQ ID NO: 4, CDR-L₂ having the amino        acid sequence of SEQ ID NO: 5 and CDR-L₃ having the amino acid        sequence of SEQ ID NO: 6.

Embodiment 2. The antibody according to Embodiment 1, wherein the aminoacid at position 8 of the CDR-H2 is selected from the group consistingof glutamine, alanine, valine, histidine, tryptophan, tyrosine, lysineand arginine, especially glutamine, histidine, tryptophan, tyrosine,lysine and arginine, in particular glutamine.

Embodiment 3. The antibody according to Embodiment 1, wherein the aminoacid at position 8 of the CDR-H2 is glutamine, histidine, arginine,tryptophan, or lysine.

Embodiment 4. The antibody according to Embodiments 1 to 3, wherein theCDR-H2 has the amino acid sequence of SEQ ID NO: 7.

Embodiment 5. The antibody according to Embodiment 1, wherein the CDR-H2has the amino acid sequence of SEQ ID NO: 8.

Embodiment 6. An antibody capable of binding to MUC1, which comprises

-   -   (i) a heavy chain variable region, which        -   (a) has an amino acid sequence which is at least 90%            identical to the amino acid sequence of SEQ ID NO: 9, and        -   (b) comprises the complementarity-determining regions (CDRs)            CDR-H1 having the amino acid sequence of SEQ ID NO: 1,            CDR-H2 having the amino acid sequence of SEQ ID NO: 2 and            CDR-H3 having the amino acid sequence of SEQ ID NO: 3, and    -   (ii) a light chain variable region, which        -   (a) has an amino acid sequence which is at least 90%            identical to the amino acid sequence of SEQ ID NO: 12, and        -   (b) comprises the complementarity-determining regions (CDRs)            CDR-L1 having the amino acid sequence of SEQ ID NO: 4,            CDR-L2 having the amino acid sequence of SEQ ID NO: 5 and            CDR-L3 having the amino acid sequence of SEQ ID NO: 6.

Embodiment 7. An antibody capable of binding to MUC1, which comprises

-   -   (i) a heavy chain variable region, which        -   (a) has an amino acid sequence which is at least 95%            identical to the amino acid sequence of SEQ ID NO: 9, and        -   (b) comprises the complementarity-determining regions (CDRs)            CDR-H1 having the amino acid sequence of SEQ ID NO: 1,            CDR-H2 having the amino acid sequence of SEQ ID NO: 2 and            CDR-H3 having the amino acid sequence of SEQ ID NO: 3, and    -   (ii) a light chain variable region, which        -   (a) has an amino acid sequence which is at least 95%            identical to the amino acid sequence of SEQ ID NO: 12, and        -   (b) comprises the complementarity-determining regions (CDRs)            CDR-L1 having the amino acid sequence of SEQ ID NO: 4,            CDR-L2 having the amino acid sequence of SEQ ID NO: 5 and            CDR-L3 having the amino acid sequence of SEQ ID NO: 6.

Embodiment 8. The antibody according to Embodiment 6 or 7, wherein theamino acid at position 8 of the CDR-H2 is selected from the groupconsisting of glutamine, alanine, valine, histidine, tryptophan,tyrosine, lysine and arginine, especially glutamine, histidine,tryptophan, tyrosine, lysine and arginine, in particular glutamine.

Embodiment 9. The antibody according to Embodiment 7 or 8, wherein theamino acid at position 8 of the CDR-H2 is glutamine, histidine,arginine, tryptophan, or lysine.

Embodiment 10. An antibody capable of binding to MUC1, which comprises

-   -   (i) a heavy chain variable region, which        -   (a) has an amino acid sequence which is at least 90%            identical to the amino acid sequence of SEQ ID NO: 10, and        -   (b) comprises the complementarity-determining regions (CDRs)            CDR-H1 having the amino acid sequence of SEQ ID NO: 1,            CDR-H2 having the amino acid sequence of SEQ ID NO: 7 and            CDR-H3 having the amino acid sequence of SEQ ID NO: 3, and    -   (ii) a light chain variable region, which        -   (a) has an amino acid sequence which is at least 90%            identical to the amino acid sequence of SEQ ID NO: 12, and        -   (b) comprises the complementarity-determining regions (CDRs)            CDR-L1 having the amino acid sequence of SEQ ID NO: 4,            CDR-L2 having the amino acid sequence of SEQ ID NO: 5 and            CDR-L3 having the amino acid sequence of SEQ ID NO: 6.

Embodiment 11. An antibody capable of binding to MUC1, which comprises

-   -   (i) a heavy chain variable region, which        -   (a) has an amino acid sequence which is at least 95%            identical to the amino acid sequence of SEQ ID NO: 10, and        -   (b) comprises the complementarity-determining regions (CDRs)            CDR-H1 having the amino acid sequence of SEQ ID NO: 1,            CDR-H2 having the amino acid sequence of SEQ ID NO: 7 and            CDR-H3 having the amino acid sequence of SEQ ID NO: 3, and    -   (ii) a light chain variable region, which        -   (a) has an amino acid sequence which is at least 95%            identical to the amino acid sequence of SEQ ID NO: 12, and        -   (b) comprises the complementarity-determining regions (CDRs)            CDR-L1 having the amino acid sequence of SEQ ID NO: 4,            CDR-L2 having the amino acid sequence of SEQ ID NO: 5 and            CDR-L3 having the amino acid sequence of SEQ ID NO: 6.

Embodiment 12. An antibody capable of binding to MUC1, which comprises

-   -   (i) a heavy chain variable region, which has the amino acid        sequence of SEQ ID NO: 9, and    -   (ii) a light chain variable region, which has the amino acid        sequence of SEQ ID NO: 12.

Embodiment 13. The antibody according to Embodiment 12, wherein theamino acid at position 57 of SEQ ID NO: 9 is selected from the groupconsisting of glutamine, alanine, valine, histidine, tryptophan,tyrosine, lysine and arginine, especially glutamine, histidine,tryptophan, tyrosine, lysine and arginine, in particular glutamine.

Embodiment 14. The antibody according to Embodiment 12, wherein theamino acid at position 57 of SEQ ID NO: 9 is glutamine, histidine,arginine, tryptophan, or lysine.

Embodiment 15. An antibody capable of binding to MUC1, which comprises

-   -   (i) a heavy chain variable region, which has the amino acid        sequence of SEQ ID NO: 10, and    -   (ii) a light chain variable region, which has the amino acid        sequence of SEQ ID NO: 12.

Embodiment 16. An antibody capable of binding to MUC1, which comprises

-   -   (i) a heavy chain variable region, which has the amino acid        sequence represented by amino acid Nos 20 to 136 of SEQ ID NO:        20 or 23, and    -   (ii) a light chain variable region, which has amino acid        sequence represented by amino acid Nos 21 to 133 of SEQ ID NO:        21.

Embodiment 17. An antibody capable of binding to MUC1, which comprises

-   -   (i) a heavy chain, which        -   (a) has an amino acid sequence which is at least 90% or at            least 95% identical to the amino acid sequence of SEQ ID NO:            15, and        -   (b) comprises the complementarity-determining regions (CDRs)            CDR-H1 having the amino acid sequence of SEQ ID NO: 1,            CDR-H2 having the amino acid sequence of SEQ ID NO: 2 or 7            and CDR-H3 having the amino acid sequence of SEQ ID NO: 3,            and    -   (ii) a light chain, which        -   (a) has an amino acid sequence which is at least 90% or at            least 95% identical to the amino acid sequence of SEQ ID NO:            16, and        -   (b) comprises the complementarity-determining regions (CDRs)            CDR-L1 having the amino acid sequence of SEQ ID NO: 4,            CDR-L2 having the amino acid sequence of SEQ ID NO: 5 and            CDR-L3 having the amino acid sequence of SEQ ID NO: 6.

Embodiment 18. An antibody capable of binding to MUC1, which comprises

-   -   (i) a heavy chain, which has the amino acid sequence of SEQ ID        NO: 15 or SEQ ID NO: 22, and    -   (ii) a light chain, which has the amino acid sequence of SEQ ID        NO: 16.

Embodiment 19. An antibody capable of binding to MUC1, which comprises

-   -   (i) a heavy chain, which        -   (a) has an amino acid sequence which is at least 90% or at            least 95% identical to the amino acid sequence of SEQ ID NO:            19, and        -   (b) comprises the complementarity-determining regions (CDRs)            CDR-H1 having the amino acid sequence of SEQ ID NO: 1,            CDR-H2 having the amino acid sequence of SEQ ID NO: 8 and            CDR-H3 having the amino acid sequence of SEQ ID NO: 3, and    -   (ii) a light chain, which        -   (a) has an amino acid sequence which is at least 90% or 95%            identical to the amino acid sequence of SEQ ID NO: 16, and        -   (b) comprises the complementarity-determining regions (CDRs)            CDR-L1 having the amino acid sequence of SEQ ID NO: 4,            CDR-L2 having the amino acid sequence of SEQ ID NO: 5 and            CDR-L3 having the amino acid sequence of SEQ ID NO: 6.

Embodiment 20. An antibody capable of binding to MUC1, which comprises

-   -   (i) a heavy chain, which has the amino acid sequence of SEQ ID        NO: 19, and    -   (ii) a light chain, which has amino acid sequence of SEQ ID NO:        16.

Embodiment 21. The antibody according to any one of Embodiments 1 to 20,wherein the antibody comprises at least one heavy chain, comprising theheavy chain variable region, a CH1 domain, a hinge region, a CH2 domainand a CH3 domain.

Embodiment 22. The antibody according to any one of Embodiments 1 to 20,wherein the antibody comprises two heavy chains, each comprising theheavy chain variable region, a CH1 domain, a hinge region, a CH2 domainand a CH3 domain.

Embodiment 23. The antibody according to Embodiment 21 or 22, whereinthe antibody is an IgG-type antibody, in particular an IgG1, IgG2 orIgG4-type antibody.

Embodiment 24. The antibody according to any one of Embodiments 1 to 23,wherein the antibody comprises at least one light chain, comprising thelight chain variable region and a CL domain.

Embodiment 25. The antibody according to any one of Embodiments 1 to 23,wherein the antibody comprises two light chains, each comprising thelight chain variable region and a CL domain.

Embodiment 26. The antibody according to Embodiment 24 or 25, whereinthe light chain is a κ-type light chain.

Embodiment 27. The antibody according to any one of Embodiments 1 to 26,wherein the antibody does not comprise an N-glycosylation site in theCH2 domain.

Embodiment 28. The antibody according to any one of Embodiments 1 to 26,wherein the antibody comprises an N-glycosylation site in the CH2 domainof the antibody heavy chains.

Embodiment 29. The antibody according to Embodiment 28, wherein theantibody has a glycosylation pattern having one or more of the followingcharacteristics

-   -   (i) a relative amount of glycans carrying a bisecting GlcNAc        residue of at least 0.5% of the total amount of glycans attached        to the glycosylation sites of the antibody in a composition;    -   (ii) a relative amount of glycans carrying at least one        galactose residue of at least 30% of the total amount of glycans        attached to the glycosylation sites of the antibody in a        composition;    -   (iii) a relative amount of glycans carrying a core fucose        residue of at least 60% of the total amount of glycans attached        to the glycosylation sites the antibody in a composition.

Embodiment 30. The antibody according to Embodiment 28, wherein theantibody has a glycosylation pattern having one or more of the followingcharacteristics

-   -   (i) a relative amount of glycans carrying a bisecting GlcNAc        residue of at least 0.5% of the total amount of glycans attached        to the glycosylation sites of the antibody in a composition;    -   (ii) a relative amount of glycans carrying at least one        galactose residue of at least 30% of the total amount of glycans        attached to the glycosylation sites of the antibody in a        composition;    -   (iii) a relative amount of glycans carrying a core fucose        residue of 40% or less of the total amount of glycans attached        to the glycosylation sites of the antibody in a composition.

In the following, specific embodiments of the conjugate according to thepresent invention are described.

Embodiment 31. The antibody according to any one of Embodiments 1 to 30,comprising a further agent, preferably cytotoxic agent, conjugatedthereto.

Embodiment 32. The antibody according to Embodiment 31, wherein thecytotoxic agent is a chemotherapeutic agent which is coupled to theantibody.

Embodiment 33. The antibody according to Embodiment 32, wherein thechemotherapeutic agent is selected from the group consisting of amicrotubule inhibitor such as maytansinoid, a topoisomerase I inhibitor,a DNA damaging agent, a DNA alkylating agent and a DNA minor groovebinder.

Embodiment 34. The antibody according to Embodiment 32, wherein thechemotherapeutic agent is selected from the group consisting ofmaytansinol, N^(2′)-deacetyl-N^(2′)-(3-mercapto-1-oxopropyl)-maytansine(DM1), N^(2′)-deacetyl-N^(2′)-(4-mercapto-1-oxopentyl)-maytansine (DM3),and N^(2′)-deacetyl-N^(2′)-(4-methyl-4-mercapto-1-oxopentyl)-maytansine(DM4).

Embodiment 35. The antibody according to Embodiment 32, wherein thechemotherapeutic agent is selected from the group consisting ofpyrrolobenzodiazepine (PBD), pyrrolobenzodiazepine dimer (PBD dimer),duocarmycin, duocarmycin-hydroxybenzamide-azaindole (DU BA),seco-duocarmycin-hydroxybenzamide-azaindole (seco-DUBA) and doxorubicin.

Embodiment 36. The antibody according to Embodiment 32, wherein thechemotherapeutic agent is selected from the group consisting ofindolinobenzodiazepine and oxazolidinobenzodiazepine.

Embodiment 37. The antibody according to Embodiment 32, wherein thechemotherapeutic agent is calicheamicin.

Embodiment 38. The antibody according to Embodiment 32, wherein thechemotherapeutic agent is selected from the group consisting ofcamptothecin, 7-ethyl-10-hydroxy-camptothecin (SN-38),(S)-9-dimethylaminomethyl-10-hydroxycamptothecin (topotecan),(1S,9S)-1-amino-9-ethyl-5-fluoro-1,2,3,9,12,15-hexahydro-9-hydroxy-4-methyl-10H,13H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinoline-10,13-dione(Exatecan (DX-8951)) and DXd.

Embodiment 39. The antibody according to Embodiment 32, wherein thechemotherapeutic agent is an antitumor compound represented by thefollowing formula:

Embodiment 40. The antibody according to Embodiment 32, wherein thechemotherapeutic agent is an antitumor compound represented by thefollowing formula:

Embodiment 41. The antibody according to Embodiment 31, whereinadditionally a further agent being a polypeptide or protein is fused toa polypeptide chain of the antibody.

Embodiment 42. The antibody according to Embodiment 41, wherein theantibody comprises two antibody heavy chains and two antibody lightchains and a further agent being a polypeptide or protein is fused toeach of the C termini of said antibody heavy chains or to each of the Ctermini of said antibody light chains.

Embodiment 43. The antibody according to Embodiment 41 or 42, whereinthe further agent is selected from the group consisting of cytokines,chemokines, other antibodies, antigen binding fragments, enzymes andbinding domains.

Embodiment 44. The antibody according to Embodiment 42, wherein thefurther agent is a scFv fragment specifically binding to CD3, and one ofsaid further agent is fused to the C terminus of each antibody heavychain.

Embodiment 45. The antibody according to Embodiment 42, wherein thefurther agent is a scFv fragment specifically binding to PDL1, and oneof said further agent is fused to the C terminus of each antibody lightchain.

Embodiment 46. The antibody according to Embodiments 31 to 45, whereinthe cytotoxic agent, preferably topoisomerase I inhibitor such asDX-8951 or DXd is conjugated thereto via a linker having any structureselected from the group consisting of the following formulas (a) to (f):-(Succinimid-3-yl-N)—CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂—C(═O)—,  (a)-(Succinimid-3-yl-N)—CH₂CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂—C(═O)—,  (b)-(Succinimid-3-yl-N)—CH₂CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂—O—CH₂—C(═O)—,  (c)-(Succinimid-3-yl-N)—CH₂CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—O—CH₂—C(═O)—,  (d)-(Succinimid-3-yl-N)—CH₂CH₂—C(═O)—NH—CH₂CH₂O—CH₂CH₂O—CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂—C(═O)—,and  (e)-(Succinimid-3-yl-N)—CH₂CH₂—C(═O)—NH—CH₂CH₂O—CH₂CH₂O—CH₂CH₂O—CH₂CH₂O—CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂—C(═O)—,  (f)

wherein the antibody is connected to the terminus of-(Succinimid-3-yl-N), the antitumor compound is connected to thecarbonyl group of the —(CH₂)n²-C(═O)— moiety (n² represents an integerof 1 or 3) in the rightmost of formulas (a) to (f) with the nitrogenatom of the amino group at position 1 as a connecting position, GGFGrepresents an amino acid sequence consisting ofglycine-glycine-phenylalanine-glycine linked through peptide bonds, and

-   -   -(Succinimid-3-yl-N)— has a structure represented by the        following formula:

which is connected to the antibody at position 3 thereof and isconnected to a methylene group in the linker structure containing thisstructure on the nitrogen atom at position 1.

Embodiment 47. A conjugate comprising the antibody according to any oneof Embodiments 1 to 30 conjugated to a cytotoxic agent.

Embodiment 48. The conjugate according to Embodiment 47, wherein thecytotoxic agent is a chemotherapeutic agent.

Embodiment 49. The conjugate according to Embodiment 48, wherein thechemotherapeutic agent is selected from the group consisting of amicrotubule inhibitor, a topoisomerase I inhibitor, a DNA damagingagent, a DNA alkylating agent and a DNA minor groove binder.

Embodiment 50. The conjugate according to Embodiment 48, wherein thechemotherapeutic agent is selected from the group consisting ofmaytansinol, N2′-deacetyl-N2′-(3-mercapto-1-oxopropyl)-maytansine (DM1),N2′-deacetyl-N2′-(4-mercapto-1-oxopentyl)-maytansine (DM3), andN2′-deacetyl-N2′-(4-methyl-4-mercapto-1-oxopentyl)-maytansine (DM4).

Embodiment 51. The conjugate according to Embodiment 48, wherein thechemotherapeutic agent is selected from the group consisting ofpyrrolobenzodiazepine (PBD), pyrrolobenzodiazepine dimer (PBD dimer),duocarmycin, duocarmycin-hydroxybenzamide-azaindole (DUBA),seco-duocarmycin-hydroxybenzamide-azaindole (seco-DUBA) and doxorubicin.

Embodiment 52. The conjugate according to Embodiment 48, wherein thechemotherapeutic agent is selected from the group consisting ofindolinobenzodiazepine and oxazolidinobenzodiazepine.

Embodiment 53. The conjugate according to Embodiment 48, wherein thechemotherapeutic agent is calicheamicin.

Embodiment 54. The conjugate according to Embodiment 48, wherein thechemotherapeutic agent is selected from the group consisting ofcamptothecin, 7-ethyl-10-hydroxy-camptothecin (SN-38),(S)-9-dimethylaminomethyl-10-hydroxycamptothecin (topotecan),(1S,9S)-1-amino-9-ethyl-5-fluoro-1,2,3,9,12,15-hexahydro-9-hydroxy-4-methyl-10H,13H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinoline-10,13-dione(Exatecan (DX-8951)) and DXd.

Embodiment 55. The conjugate according to Embodiment 48, wherein thechemotherapeutic agent is an antitumor compound represented by thefollowing formula:

Embodiment 56. The conjugate according to Embodiment 48, wherein thechemotherapeutic agent is an antitumor compound represented by thefollowing formula:

Embodiment 57. The conjugate according to Embodiment 47, wherein afurther agent being a polypeptide or protein is fused to a polypeptidechain of the antibody.

Embodiment 58. The conjugate according to Embodiment 57, wherein theantibody comprises two antibody heavy chains and two antibody lightchains and a further agent being a polypeptide or protein is fused toeach of the C termini of said antibody heavy chains or to each of the Ctermini of said antibody light chains.

Embodiment 59. The conjugate according to Embodiment 57 or 58, whereinthe further agent is selected from the group consisting of cytokines,chemokines, other antibodies, antigen binding fragments, enzymes andbinding domains.

Embodiment 60. The conjugate according to Embodiment 58, wherein thefurther agent is a scFv fragment specifically binding to CD3, and one ofsaid further agent is fused to the C terminus of each antibody heavychain.

Embodiment 61. The conjugate according to Embodiment 58, wherein thefurther agent is a scFv fragment specifically binding to PDL1, and oneof said further agent is fused to the C terminus of each antibody lightchain.

Embodiment 62. The conjugate according to any one of Embodiments 47 to61, wherein the antibody is conjugated to further agent orchemotherapeutic agent, preferably topoisomerase I inhibitor such asDX-8951 or DXd via a linker having any structure selected from the groupconsisting of the following formulas (a) to (f):-(Succinimid-3-yl-N)—CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂—C(═O)—,  (a)-(Succinimid-3-yl-N)—CH₂CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂—C(═O)—,  (b)-(Succinimid-3-yl-N)—CH₂CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂—O—CH₂—C(═O)—,  (c)-(Succinimid-3-yl-N)—CH₂CH₂CH₂CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂—O—CH₂—C(═O)—,  (d)-(Succinimid-3-yl-N)—CH₂CH₂—C(═O)—NH—CH₂CH₂O—CH₂CH₂O—CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂—C(═O)—,and  (e)-(Succinimid-3-yl-N)—CH₂CH₂—C(═O)—NH—CH₂CH₂O—CH₂CH₂O—CH₂CH₂O—CH₂CH₂O—CH₂CH₂—C(═O)-GGFG-NH—CH₂CH₂CH₂—C(═O)—,  (f)

wherein the antibody is connected to the terminus of-(Succinimid-3-yl-N), the antitumor compound is connected to thecarbonyl group of the —(CH₂)n²-C(═O)— moiety (n² represents an integerof 1 or 3) in the rightmost of formulas (a) to (f) with the nitrogenatom of the amino group at position 1 as a connecting position, GGFGrepresents an amino acid sequence consisting ofglycine-glycine-phenylalanine-glycine linked through peptide bonds, and

-   -   -(Succinimid-3-yl-N)— has a structure represented by the        following formula:

which is connected to the antibody at position 3 thereof and isconnected to a methylene group in the linker structure containing thisstructure on the nitrogen atom at position 1.

Embodiment 63. The conjugate according to any one of Embodiments 47 to61, wherein the antibody or the antigen binding fragment thereof isconjugated to a drug linker represented by the following formula(wherein asterisk* represents the point of connection to the antibody)by a thioether bond, the antibody comprises any one of the followingcombinations a) to f) of a heavy chain variable region and a light chainvariable region or heavy chain and light chain:

-   -   (a) the heavy chain variable region has the amino acid sequence        of SEQ ID NO: 10 and the light chain variable region has the        amino acid sequence of SEQ ID NO: 12,    -   (b) the heavy chain variable region has the amino acid sequence        of SEQ ID NO: 11 and the light chain variable region has the        amino acid sequence of SEQ ID NO: 12,    -   (c) the heavy chain comprising the amino acid sequence of SEQ ID        NO: 15 or 22 and the light chain comprising the amino acid        sequence of SEQ ID NO: 16,    -   (d) the heavy chain comprising the amino acid sequence of SEQ ID        NO: 19 and the light chain comprising the amino acid sequence of        SEQ ID NO: 16.    -   (e) the heavy chain having an amino acid sequence represented by        amino acid Nos 1 to 446 of SEQ ID NO: 15 or 22 and the light        chain having an amino acid sequence represented by amino acid        Nos 1 to 219 of SEQ ID NO: 16, and    -   (f) the heavy chain having an amino acid sequence represented by        amino acid Nos 1 to 446 of SEQ ID NO: 19 and the light chain        having an amino acid sequence represented by amino acid Nos 1 to        219 of SEQ ID NO: 16:

Embodiment 64. A conjugate or an antibody, which is represented by thefollowing formula,

wherein AB represents the antibody, the antibody comprises the heavychain variable region having the amino acid sequence of SEQ ID NO: 10and the light chain variable region having the amino acid sequence ofSEQ ID NO: 12, y represents an average number of units of thedrug-linker structure conjugated to the antibody per itself and y is ina range of from 1 to 10, in a range of from 2 to 8, in a range of from 3to 8, in a range of from 7 to 8 or in a range of from 7.5 to 8, and theantibody is conjugated to a drug linker structure represented by theabove formula by a thioether bond.

Embodiment 65. A conjugate or an antibody, which is represented by thefollowing formula,

wherein AB represents the antibody, the antibody comprises the heavychain variable region having the amino acid sequence of SEQ ID NO: 11and the light chain variable region having the amino acid sequence ofSEQ ID NO: 12, y represents an average number of units of thedrug-linker structure conjugated to the antibody per itself and y is ina range of from 1 to 10, in a range of from 2 to 8, in a range of from 3to 8, in a range of from 7 to 8 or in a range of from 7.5 to 8, and theantibody is conjugated to a drug linker structure represented by theabove formula by a thioether bond.

Embodiment 66. A conjugate or an antibody, which is represented by thefollowing formula,

wherein AB represents the antibody, the antibody comprises the heavychain having the amino acid sequence of SEQ ID NO: 15 or 22 and thelight chain having the amino acid sequence of SEQ ID NO: 16, yrepresents an average number of units of the drug-linker structureconjugated to the antibody per itself and y is in a range of from 1 to10, in a range of from 2 to 8, in a range of from 3 to 8, in a range offrom 7 to 8 or in a range of from 7.5 to 8, and the antibody isconjugated to a drug linker structure represented by the above formulaby a thioether bond.

Embodiment 67. A conjugate or an antibody, which is represented by thefollowing formula,

wherein AB represents the antibody, the antibody comprises the heavychain having the amino acid sequence of SEQ ID NO: 19 and the lightchain having the amino acid sequence of SEQ ID NO: 16, y represents anaverage number of units of the drug-linker structure conjugated to theantibody per itself and y is in a range of from 1 to 10, in a range offrom 2 to 8, in a range of from 3 to 8, in a range of from 7 to 8 or ina range of from 7.5 to 8, and the antibody is conjugated to a druglinker structure represented by the above formula by a thioether bond.

Embodiment 68. The conjugate or the antibody according to any one ofEmbodiments 31 to 67, wherein the antibody comprises one or moremodifications selected from the group consisting of defucosylation,reduced fucose, N-linked glycosylation, O-linked glycosylation,N-terminal processing, C-terminal processing, deamidation, isomerizationof aspartic acid, oxidation of methionine, the substitutions of twoleucine (L) residues to alanine (A) at position 234 and 235 of the heavychain (LALA), amidation of a proline residue, and a deletion or lack ofone or two amino acids at the carboxyl terminus.

Embodiment 69. The conjugate or the antibody according to Embodiment 68,wherein the antibody comprises a deletion or lack of one or two aminoacid(s) in the carboxyl terminus of the heavy chain.

Embodiment 70. The conjugate or the antibody according to Embodiment 69,wherein the antibody comprises two heavy chains, both of which lack onecarboxyl-terminal amino acid residue.

Embodiment 71. The conjugate or the antibody, which is represented bythe following formula,

wherein AB represents the antibody, the antibody comprises the heavychain having an amino acid sequence represented by amino acid Nos 1 to446 of SEQ ID NO: 15 or 22 and the light chain having an amino acidsequence represented by amino acid Nos 1 to 219 of SEQ ID NO: 16, yrepresents an average number of units of the drug-linker structureconjugated to the antibody per itself and y is in the range of 1 to 10,in a range of from 2 to 8, in a range of from 3 to 8, in a range of from7 to 8 or in a range of from 7.5 to 8, and the antibody is conjugated toa drug linker structure represented by the above formula by a thioetherbond.

Embodiment 72. The conjugate or the antibody, which is represented bythe following formula,

wherein AB represents the antibody, the antibody comprises the heavychain having an amino acid sequence represented by amino acid Nos 1 to446 of SEQ ID NO: 19 and the light chain having an amino acid sequencerepresented by amino acid Nos 1 to 219 of SEQ ID NO: 16, y represents anaverage number of units of the drug-linker structure conjugated to theantibody per itself and y is in the range of 1 to 10, in a range of from2 to 8, in a range of from 3 to 8, in a range of from 7 to 8 or in arange of from 7.5 to 8, and the antibody is conjugated to a drug linkerstructure represented by the above formula by a thioether bond.

Embodiment 73. The conjugate or the antibody according to any one ofEmbodiments 31 to 72, wherein the number of conjugated drug moleculesper antibody molecule is 8.

Embodiment 74. A pharmaceutical composition comprising the antibody orthe conjugate according to any one of Embodiments 31 to 73 and one ormore further components selected from the group consisting of solvents,diluents, and excipients.

Embodiment 75. The antibody or conjugate according to any one ofEmbodiments 31 to 73 or the pharmaceutical composition according toEmbodiment 74 for use in medicine.

Embodiment 76. The antibody or conjugate according to any one ofEmbodiments 31 to 73 or the pharmaceutical composition according toEmbodiment 74 for use in the treatment, prognosis, diagnosis and/ormonitoring of diseases associated with abnormal cell growth such ascancer; infections such as bacterial, viral, fungal or parasiticinfections; inflammatory diseases such as autoimmune diseases andinflammatory bowel diseases; and diseases associated with a reduceimmune activity such as immunodeficiencies.

Embodiment 77. The antibody, conjugate or pharmaceutical compositionaccording to Embodiment 76 for use in the treatment of cancer, inparticular cancer expressing TA-MUC1, wherein the cancer is selectedfrom the group consisting of cancer of ovarian cancer, breast cancer,pancreatic cancer, lung cancer, colon cancer, stomach cancer, livercancer, kidney cancer, blood cancer, endometrial cancer, thyroid cancer,leukemia, seminomas, melanomas, carcinomas, teratomas, lymphomas,sarcomas, mesotheliomas, neuroblastomas, gliomas, rectal cancer, adrenalcancer, skin cancer, cancer of the brain, cervical cancer, intestinalcancer, intestine cancer, head and neck cancer, gastrointestinal cancer,lymph node cancer, esophagus cancer, colorectal cancer, ear, nose andthroat (ENT) cancer, prostate cancer, bladder cancer, cancer of theuterus and the metastases thereof.

Embodiment 78. The antibody, conjugate or pharmaceutical compositionaccording to Embodiment 76 for use in the treatment of infections,wherein the infection is selected from the group consisting of bacterialinfections, viral infections, fungal infections and parasiticinfections.

Embodiment 79. The antibody, conjugate or pharmaceutical compositionaccording to Embodiment 76 for use in the treatment of autoimmunediseases, wherein the autoimmune disease is selected from the groupconsisting of celiac disease, diabetes mellitus type 1, Graves disease,inflammatory bowel disease, multiple sclerosis, psoriasis, rheumatoidarthritis and systemic lupus erythematosus.

Embodiment 80. A method for treating cancer in a subject in need thereofcomprising, administering to the subject with cancer, in particularcancer expressing TA-MUC1 a therapeutically effective amount of theconjugate or the antibody according to any one of Embodiments 31 to 73or the composition according to Embodiment 74.

Embodiment 81. The method for treating cancer according to Embodiment80, wherein the cancer is selected from the group consisting of ovariancancer, breast cancer, pancreatic cancer, lung cancer, colon cancer,stomach cancer, liver cancer, kidney cancer, blood cancer, endometrialcancer, thyroid cancer, leukemia, seminomas, melanomas, carcinomas,teratomas, lymphomas, sarcomas, mesotheliomas, neuroblastomas, gliomas,rectal cancer, adrenal cancer, skin cancer, cancer of the brain,cervical cancer, intestinal cancer, intestine cancer, head and neckcancer, gastrointestinal cancer, lymph node cancer, esophagus cancer,colorectal cancer, ear, nose and throat (ENT) cancer, prostate cancer,bladder cancer, cancer of the uterus and the metastases thereof.

FIGURES

FIG. 1 shows ELISA binding curves of the anti-MUC1 antibodies todifferent MUC1 peptides. (A) shows antigen binding of PankoMab N54Q(PM-N54Q) lacking Fab glycosylation and PankoMab comprising Fabglycosylation (PM) to the MUC1 peptide comprising the epitope sequencePDTR. The threonine of the MUC1 peptide is glycosylated with Tn, sTn, TFor sTF. (B) shows binding of PankoMab and PM-N54Q to the MUC1 peptidecomprising the epitope sequence variant PESR. The serine of the MUC1peptide is glycosylated with Tn. (C) shows binding of PM-N54Q to theMUC1 peptide comprising the epitope sequence PDTR. The threonine of theMUC1 peptide is glycosylated with Tn or not glycosylated. (D) showsbinding of several N54X variants to Tn-PDTR MUC1 peptide compared toPankoMab comprising Fab glycosylation diluted from cell culturesupernatant of transiently transfected cells. (E) shows binding curvesof three purified N54X variants without Fab glycosylation in comparisonto PankoMab with Fab glycosylation on Tn-PDTR, TF-PDTR andnon-glycosylated PDTR MUC1 peptide. (F) shows binding of two frameworkvariants of PM-N54Q to Tn-PDTR MUC1 peptide compared to PankoMab withFab glycosylation. For framework variant mf-a nine amino acids aremutated in the VH and three in the VL framework, for mf-b also nineamino acids are mutated in the VH and four in the VL framework.

FIG. 2 shows surface plasmon resonance (Biacore) binding of theanti-MUC1 antibodies PM and PM-N54Q to a glycosylated PDTR-MUC1 peptide.The maximal binding signal of different concentrations of PM-N54Q andPankoMab are plotted against the antibody concentration.

FIG. 3 shows results of Fluorescence Proximity Sensing on DRXinstrument. Association and dissociation curves are shown. (A) PM withFab glycosylation compared to (B) PM-N54Q without Fab glycosylation

FIG. 4 shows an SDS acrylamide gel of an electrophoretic separation ofPM-N54Q and PankoMab under non-reducing (left) and reducing (right)conditions. Lane 1: PM-N54Q after capture step; lane 2: PM-N54Q afterpolishing step; lane 3: PankoMab after capture step; lane 4: PankoMabafter polishing step; lane 5: molecular weight marker.

FIG. 5 shows the Coomassie blue stained gel of an isoelectric focusingassay with PM-N54Q lacking Fab glycosylation and PankoMab beingFab-glycosylated. Lane 1: PankoMab with Fab glycosylation; lane 2:PM-N54Q without Fab glycosylation.

FIG. 6 shows anti-MUC1 antibody binding to Fcγ receptor IIIa. Increasingconcentrations of the antibody PM-N54Q or PankoMab displacerabbit-anti-mouse coupled acceptor beads from FcγRIIIa loaded donorbeads, thereby reducing the detected chemiluminescence. In FIG. 6Alow-fucosylated antibodies and in FIG. 6B high-fucosylated antibodieswere applied into the assay.

FIG. 7 shows binding of the anti-MUC1 antibodies PM-N54Q, PM-N54D and PMwith Fab glycosylation to the tumor cell lines (A) CaOV-3 and (B) HSC-4as analyzed by flow cytometry.

FIG. 8 shows cytotoxic activity of A) control hIgG-ADC, naked PankoMaband PankoMab-ADC against cancer cell lines MDA-MB-468 with expression ofTA-MUC1 proteins, B) control hIgG-ADC, naked PankoMab and PankoMab-ADCagainst cancer cell lines HCT-15 without expression of TA-MUC1 proteins,C) control hIgG-ADC, naked PankoMab, PankoMab-ADC, naked PM-N54Q andPM-N54Q-ADC against cancer cell lines NCI-H441 with expression ofTA-MUC1 proteins, D) control hIgG-ADC, naked PankoMab PankoMab-ADC,naked PM-N54Q and PM-N54Q-ADC against cancer cell lines HPAC withexpression of TA-MUC1 proteins. The cells were treated with eachcompound for 6 days and cell viability (%) was calculated by ATP assay.Data represent the mean±SD (N=3).

FIG. 9 shows antitumor efficacy of control hIgG-ADC, naked PankoMab andPankoMab-ADC against MDA-MB-468-bearing nude mice. Control hIgG-ADC,naked PankoMab and PankoMab-ADC at doses of 3 mg/kg or vehicle (acetatebuffer solution) was single dose administered intravenously intoMDA-MB-468-bearing nude mice (N=6/group). Data of estimated tumor volumerepresent the mean±SEM. The arrow shows the timing of administration.Estimated tumor volumes at 21 days after the administration of thePankoMab-ADC were compared with that of control hIgG-ADC or that ofnaked PankoMab treated group by Student t-test. ***P<0.001.

FIG. 10 shows antitumor efficacy of control hIgG-ADC, naked PankoMab andPankoMab-ADC against HCC70-bearing nude mice. Control hIgG-ADC, nakedPankoMab and PankoMab-ADC at doses of 10 mg/kg or vehicle (acetatebuffer solution) was single dose administered intravenously intoHCC70-bearing nude mice (N=6/group). Data of estimated tumor volumerepresent the mean±SEM. The arrow shows the timing of administration.Estimated tumor volumes at 21 days after the administration of thePankoMab-ADC were compared with that of the control hIgG-ADC or nakedPankoMab treated group by Student t-test. ***P<0.001.

FIG. 11 shows antitumor efficacy of control hIgG-ADC, naked PM-N54Q,PankoMab-ADC and PM-N54Q-ADC against HPAC-bearing nude mice. ControlhIgG-ADC, naked PM-N54Q, PankoMab-ADC and PM-N54Q-ADC at doses of 10mg/kg or vehicle (acetate buffer solution) was single dose administeredintravenously into HPAC-bearing nude mice (N=6/group). Data of estimatedtumor volume represent the mean±SEM. The arrow shows the timing ofadministration. Estimated tumor volumes at 21 days after theadministration of PankoMab-ADC and PM-N54Q-ADC were compared with thatof the control hIgG-ADC treated group by Dunnett's test. ***P<0.001.

FIG. 12 shows antitumor efficacy of control hIgG-ADC, naked PankoMab,naked PM-N54Q, PankoMab-ADC and PM-N54Q-ADC against NCI-H441-bearingnude mice. Naked PankoMab, naked PM-N54Q at doses of 10 mg/kg, controlhIgG-ADC, PankoMab-ADC and PM-N54Q-ADC at doses of 3 mg/kg or vehicle(acetate buffer solution) was single dose administered intravenouslyinto NCI-H441-bearing nude mice (N=6/group). Data of estimated tumorvolume represent the mean±SEM. The arrow shows the timing ofadministration. Estimated tumor volumes at 31 days after theadministration of PankoMab-ADC and PM-N54Q-ADC were compared with thatof the control hIgG-ADC treated group by Dunnett's test. ***P<0.001.

FIG. 13 shows antitumor efficacy of control hIgG-ADC, naked PankoMab,naked PM-N54Q, PankoMab-ADC and PM-N54Q-ADC against OVCAR-5-bearing nudemice. Control hIgG-ADC, naked PankoMab, naked PM-N54Q, PankoMab-ADC andPM-N54Q-ADC at doses of 10 mg/kg or vehicle (acetate buffer solution)was single dose administered intravenously into OVCAR-5-bearing nudemice (N=6/group). Data of estimated tumor volume represent the mean±SEM.The arrow shows the timing of administration. Estimated tumor volumes at14 days after the administration of PankoMab-ADC and PM-N54Q-ADC werecompared with that of the control hIgG-ADC treated group by Dunnett'stest. Estimated tumor volumes at 14 days after the administration ofPM-N54Q-ADC were compared with that of PankoMab-ADC treated group byStudent t-test. ***P<0.001.

FIG. 14 shows antitumor efficacy of control hIgG-ADC, PankoMab-ADC andPM-N54Q-ADC against HCT-15-bearing nude mice. Control hIgG-ADC,PankoMab-ADC and PM-N54Q-ADC at doses of 10 mg/kg or vehicle (acetatebuffer solution) was single dose administered intravenously intoHCT-15-bearing nude mice (N=6/group). Data of estimated tumor volumerepresent the mean±SEM. The arrow shows the timing of administration.Estimated tumor volumes at 21 days after the administration ofPankoMab-ADC and PM-N54Q-ADC were compared with that of control hIgG-ADCtreated group by Dunnett's test. ***P<0.001.

FIG. 15 shows the amino acid sequence of the heavy chain of thehumanized antibody PM N54Q (SEQ ID No: 15, wherein the amino acid atposition 57 is Gln, namely SEQ ID No: 22).

FIG. 16 shows the amino acid sequence of the light chain of thehumanized antibodies PM N54Q and PankoMab (SEQ ID No: 16).

FIG. 17 shows the amino acid sequence of the heavy chain of thehumanized antibody PankoMab (SEQ ID No: 19).

FIG. 18 shows the amino acid sequence of the heavy chain of chimericantibody PM N54Q (SEQ ID No: 20, wherein the amino acid at position 76is Gln, namely SEQ ID No: 23).

FIG. 19 shows the amino acid sequence of the light chain of chimericantibody PM N54Q (SEQ ID No: 21).

EXAMPLES Example 1

1. Production of Anti-MUC1 Antibodies

The nucleic acid sequence of the heavy chain of humanized PankoMabantibody (see, e.g., WO 2011/012309) was modified by mutating the codonfor Asn54 according to the Kabat/Eu numbering system (amino acidposition 57 in SEQ ID NO: 11) into the codon for any amino acid exceptAsn, especially for Gln.

1) Production of the Anti-MUC1 Antibodies in a Human Myeloid LeukemiaDerived Cell Line

Vectors comprising the coding sequences of the γ1-type heavy chain andthe κ-type light chain of the mutated antibodies were transfected intothe human myeloid leukemia derived cell line NM-H9D8 (DSM ACC2806). Thedifferent αMUC1-antibodies comprising the N54X mutation (PankoMabN54X/PM-N54X, wherein X is any amino acid except N/Asn) or amino acidmutations in the framework sequences of the VH and VL were expressed inthe obtained clones, producing the constructs with a human glycosylationpattern. The concentration of the αMUC1-antibodies in the supernatantwas determined by Octet measurement using Protein A coated pins or werequantified by UV280 absorbance after purification by protein Achromatography. The binding characteristics of the differentαMUC1-antibodies were determined by Antigen-ELISA (see example 2), andselected purified antibodies were also analyzed by Scatchard analysis(see example 3), by Biacore (see example 4a), by DRX² switchSENSE®Technology (see example 4b), or by flow cytometry (example 7).

In addition, PM-N54Q and non-mutated PankoMab with Fab-glycosylationwere also expressed in the human myeloid leukemia derived cell lineNM-H9D8-E6Q12 (DSM ACC2856) expressing antibody with reduced fucose.Together with the same antibodies expressed in NM-H9D8, these antibodieswere purified and analyzed in example 6 for their binding behavior to Fcgamma receptor III A.

2) Production of the Anti-MUC1 Antibody in CHO Cell Line

PM-N54Q encoding sequences (nucleotide sequence of heavy chain ofPM-N54Q represented by SEQ ID NO: 17 and nucleotide sequence of lightchain of PM-N54Q) represented by SEQ ID NO: 18) which was synthesized byGeneArt™ of ThermoFisher scientific were cloned into expression vectorsand resulting plasmids were electro-transfected into CHO cells. Pooledcells grown under selection pressure were applied to manufacture PM-N54Qmutant antibody with general procedures. Anti-MUC 1 antibody (PM-N54Q)produced in CHO cell line was used for Example 8 and 9.

2. PankoMab-ADC, N54Q-ADC and DXd

PankoMab-GEX, which refers to a humanized, anti-TA-MUC1 monoclonalantibody comprising a glycosylation site in CDR2-H2 (Fab glycosylation),and PM-N54Q, which refers to a humanized anti-TA-MUC1 monoclonal lackingFab glycosylation (Example 1-1), PankoMab-ADC and PM-N54Q-ADC wereproduced by a known method such as WO 2014/057687 and WO 2015/115091.PankoMab-GEX antibody comprises a heavy chain comprising SEQ ID NO: 19and a light chain comprising SEQ ID NO: 16, thus the PankoMab-GEXantibody being linked to a drug-linker of Formula 2. PM-N54Q antibodymentioned above comprises a heavy chain comprising SEQ ID NO: 15 and alight chain comprising SEQ ID NO: 16, thus the PM-N54Q antibody beinglinked to a drug-linker of Formula 2.

Such PankoMab-ADC and PM-N54Q-ADC structures show the following Formula5 (y: The number of conjugated drug molecules per antibody molecule isfrom 4 to 8, namely, average number of conjugated drug molecules (y) perantibody: approximately 8), AB represents PankoMab or PM-N54Q.

Control hIgG-ADC was composed of a humanized IgG1 isotype controlmonoclonal antibody, not binding to mammalian cells, and the samedrug-linker as PnakoMab-ADC and PM-N54Q-ADC. ADC payload (DXd) wasproduced by a known method such as WO 2014/057687 and WO 2015/115091.

Example 2: Antigen ELISA

The antigen binding characteristics of PankoMab N54X, wherein theN-glycosylation site in the Fab part is knocked out, was compared toPankoMab having an N-glycosylation site in its Fab part.

Binding characteristics of the Fab-deglycosylated version of theMUC1-specific antibody PankoMab (PM-N54Q) compared to the (glycosylated)PankoMab-GEX® were analyzed using differently glycosylated and thenon-glycosylated MUC1-derived tandem repeat peptides in ELISA studies.In principle, both antibodies show the same gradation by means ofbinding to glycosylated PDTR peptides(APPAHGVTSAPD-T(X)-RPAPGSTAPPAHGVTSA) (SEQ ID NO: 24) with differentglycosylations at T: Strongest binding was observed to the PDTR peptidecarrying a Galß1-3GalNAc_(alpha) (TF) followed by sialylated TF andGalNAc_(alpha) (Tn) O-glycosylation. Binding to sialylatedGalNAc_(alpha) (sTn) O-glycosylation was significantly lower. AsPankoMab-GEX®, PM-N54Q showed only little binding affinity tonon-glycosylated MUC1 PDTR peptide indicating adequate tumor specificity(FIG. 1C).

However, in comparison to PankoMab-GEX® four-fold higher binding wasfound for PM-N54Q in the TA-MUC1 antigen ELISA using the biotinylatedglycopeptide carrying a GalNAc_(alpha) (Tn) O-glycan. PM-N54Q bindsabout seven-fold better to the same MUC1 peptide when glycosylated withsialylated GalNAc_(alpha) (sTn). The binding to Galß1-3GalNAc_(alpha)(TF) and sialylated TF (sTF) at the threonine of the PDTR-sequence (FIG.1A) was two-fold better for PM-N54Q.

Both antibodies show strongly diminished binding to the MUC1 peptidevariant APPAHGVTSAPE-S(Tn)-RPAPGSTAPPAHGVTSA (SEQ ID NO: 25) with Tnglycosylation at the serine compared to that at PDT(Tn)R-peptide.However, also here the Fab-deglycosylated PM-N54Q binds significantlystronger than PankoMab-GEX® (FIG. 1B).

Different other Fab-deglycosylated PM-N54X variants were compared toPankoMab having an N-glycosylation in its Fab part. First, all variantswere compared directly from the supernatant, without purification. Theconcentration was determined by Octet. All PM-N54X variants bound betterthan Fab-glycosylated PM. In addition, a clear trend depending on thechemical properties of the amino acid side chain was visible.

Carboxylic acid groups at the side chain showed the lowest bindingenhancement. Best binding was observed for amino acids with one or twonitrogens (as primary or secondary amines) (FIG. 1D).

In addition, selected Fab-deglycosylated variants (PM-N54H, -W, and -Q)were purified by Protein A chromatography and analyzed on ELISA (FIG.1E). The improvement of binding to TF-MUC1 peptide is about 5- to 8-foldand to Tn-MUC1 peptide about 2- to 3-fold compared to PankoMab withFab-glycosylation, respectively.

Furthermore, two different framework variants of the PM-N54Q wereanalyzed for the binding to the Tn-glycosylated PDTR-MUC1 peptide inELISA (see FIG. 1F). The framework variant mf-a carries nine amino acidmutations in the VH and three in the VL framework; the variant mf-bcarries also nine amino acid mutations in the VH and four in the VLframework. Both mutated variants show similar binding compared to thePM-N54Q antibody.

Example 3: Saturation Binding Analyses of Anti-MUC1 Antibodies to MCF-7and ZR-75-1 Cells

Two factors are especially critical for the therapeutic suitability ofan antibody: the affinity and number of binding sites of an antibody ontumor cells.

Binding of the Fab-deglycosylated version of the MUC1-specific antibodyPankoMab (PM-N54Q) on TA-MUC-1 positive human tumor cell lines wasevaluated using radiolabeled antibodies by saturated binding analysis onthe human mamma carcinoma cell lines ZR-75-1 and MCF-7 in comparison toFab-glycosylated PankoMab-GEX®. The antibodies were chelated with a12-fold molar excess of p-SCN-Benzyl-DTPA in 50 mM sodium carbonate, 150mM NaCl, pH 8.7, for 2 h at 37° C., followed by over-night incubation at2-8° C. Free chelator was removed over desalting columns and dead-endfiltration (50 kDa cut-off, 6× buffer exchange to PBS). The chelatedantibodies were radiolabeled with carrier-free ¹¹¹In (2 μCi/μg antibody)for 1 h at 37° C. in 6 mM phosphate, 1.6 mM KCl, 80 mM NaCl, 0.2 MNa-acetate, 0.1 M HCl. The preparations were neutralized by addition of8-9 fold volume of 10× concentrated PBS. About 1/50 volume of fetalbovine serum were added to the neutralized labelled antibodypreparation. Per cell binding approach 1*10⁶ cells were used. Severalconcentrations of labelled antibodies were added to the pelleted cells(30-1000 ng/200 μL in 1% BSA/PBS). The resuspended cell-antibodymixtures were measured in a gamma-counter and incubated 1 h at 4° C.Cells with bound antibodies were separated by centrifugation and washedwith 1% BSA/PBS for another hour at 4° C. The cell pellet was thenmeasured for bound ¹¹¹In-labelled antibody in a gamma counter.Evaluation was performed by “one-site specific ka” in GraphPad Prism.The obtained data are summarized in Table 1. The data show the highaffinity and very high number of binding sites of PM-N54Q on these tumorcells. The binding was more than 2.5-fold higher than for PankoMab-GEX®and also the number of binding sites was slightly increased.

TABLE 1 Association constant and antigen binding sites on MUC1⁺ tumorcells K_(ass)[1/M] ZR-75-1 MCF-7 PM w Fab glyc. 1.2 × 10⁷ 3 × 10⁷ PMN54Q w/o Fab glyc. 3.4 × 10⁷ 7.8 × 10⁷ Binding sites ZR-75-1 MCF-7 PM wFab glyc. 20 × 10⁵ 0.6 × 10⁵ PM N54Q w/o Fab glyr. 30 × 10⁵ 0.9 × 10⁵

Example 4a: Surface Plasmon Resonance (BiaCore) Analysis

Binding of the Fab-deglycosylated version of the MUC1-specific antibodyPankoMab (PM-N54Q) on TA-MUC-1 derived glycosylated peptide wasevaluated by surface plasmon resonance analysis (Biacore). Astreptavidin sensor chip was coated with biotinylated TA-MUC1 peptide(Tn glycosylated or not glycosylated). PankoMab and PM-N54Q were dilutedsequentially 1:3 from 3,600 to 4.9 nM in HPS-EP. The dilutions wereinjected at 50 μL/min. Maximal binding of each concentration wasdetermined as response units (RU), respectively, and evaluated withGraphPad Prism using “one-site specific binding”. FIG. 2 shows theobtained binding curves with PM-N54Q compared to PankoMab-GEX®.Affinities (K_(D)) of 388 nM and 652 nM were calculated for PM-N54Q andPankoMab-GEX®, respectively. Therefore, in this experimental setting anearly two-fold increase in affinity was detectable.

Example 4b: Fluorescence Proximity Sensing (by DRX², Dynamic Biosensors)

A new method to determine binding constants and affinity is thefluorescence proximity sensing using single stranded DNA (96mer) spottedon a chip and complementary DNA coupled to a ligand. In the presentstudy streptavidin was used as a ligand to capture biotinylated TA-MUC1peptides. Binding of PankoMab to the peptides resulted in a fluorescencechange. On- and off-rates can be calculated during association anddissociation. Due to a higher sensitivity faster interactions can bemonitored compared to surface plasmon resonance. This results in bindingkinetics different from SPR but more comparable to the “gold standard”method KinExA, measured in a liquid system.

PankoMab and PM-N54Q were diluted from 300 nM in 1:9 steps to 3.67 nM inPE140 buffer and applied to the chip-bound peptides. Binding curves wereevaluated by mono-exponential global fit (instrument software). Bindingcurves of PM and PM-N54Q are exemplarily shown in FIGS. 3A and B.Calculated affinities of PankoMab variants are shown in Table 2:

TABLE 2 Dissociation constants of PankoMab variants to antigen peptidePankoMab variant K_(D) PM with Fab glycosylation 4.1 nM PM-N54D 1.9 nMPM-N54Q 1.6 nM PM-N54H 0.6 nM

Example 5: Biochemical Characterization

Non-reducing and reducing SDS-PAGE is used to analyze purity andidentity of an antibody. The band pattern in non-reducing gels shows themajor band at about 160 kDa and methodical artefacts of heavy and lightchains and combinations thereof (˜25, 50-55, 75, 110, 135 kDa). Reducinggels show distinct light and heavy chain bands at and 50-55 kDa. Due tolack of the Fab glycosylation PM-N54Q has a smaller heavy chain, asexpected (see FIG. 4 , right).

The charge profile is clearly different, as shown by isoelectricfocusing (IEF; see FIG. 5 ). The Fab glycosylation is considerablysialylated, whereas the Fc glycosylation is only minimally sialylated.Thus PankoMab-GEX® has more charged isoforms than PM-N54Q, reflectingits higher level of negatively charged sialic acids in the Fab part.

Example 6: Fcγ Receptor Binding

FcγR binding assays for FcγRIIIa (CD16a) are based on the AlphaScreen®technology of PerkinElmer. The AlphaScreen® platform relies on simplebead-based technology of PerkinElmer and is a more efficient alternativeto traditional ELISA since no washing steps are necessary.

For the receptor binding assays, His-tagged FcγRIIIa (Glycotope GmbH) iscaptured by Ni-chelate donor beads. Anti-MUC1 antibodies andrabbit-anti-mouse coupled acceptor beads compete for binding to FcγR. Incase of interaction of FcγR with rabbit-anti-mouse-bound acceptor beads,donor and acceptor beads come into close proximity which leads, uponlaser excitation at 680 nm, to light emission. A maximum signal isachieved (signal_(max)) without a competitor. In case of competition,where a test antibody binds to FcγR, the signal_(max) is reduced in aconcentration-dependent manner. Chemiluminescence was quantified bymeasurement at 520-620 nm (AlphaScreen® method) using an EnSpire 2300multilabel reader (PerkinElmer). All results were expressed as themean±standard deviation of duplicate samples. The data were evaluatedand calculated using non-linear curve fitting (sigmoidal dose-responsevariable slope) with GraphPad Prism 5 software. As a result, aconcentration dependent sigmoidal curve was obtained, which is definedby top-plateau, bottom-plateau, slope and EC₅₀.

As shown in FIGS. 6A and B, the FcγRIIIa binding affinity was comparablefor PankoMab N54Q and PankoMab whereby in Figure A low-fucosylatedantibodies and in Figure B high-fucosylated antibodies were applied intothe assay. Hence, removal of the Fab glycosylation did not affectreceptor interaction of the antibody.

Example 7: Binding to Cellular TA-MUC1

N54Q and N54D were transiently expressed and purified by protein Achromatography. Binding of the two variants to cell surface TA-MUC1 wascompared to PM with Fab glycosylation using two different carcinoma celllines. The tongue squamous cell carcinoma line HSC-4 expresses TA-MUC1to a medium degree and the ovarian carcinoma cell line CaOV-3 to a highdegree. Tumor cells were incubated with antibodies in serial dilutionsand bound antibodies were detected using a Phycoerythrin-conjugated goatanti-human IgG (heavy and light chain) antibody. A human IgG control wasincluded to control for background staining. Binding was analyzed byflow cytometry.

The analyzed constructs PM, PM-N54Q and PM-N54D show strong and specificbinding to the TA-MUC1 expressing HSC-4 and CaOV-3 cells compared to ahuman IgG1 control (FIG. 7 ). The binding of PM-N54D to theTA-MUC1^(high) CaOV-3 cells was comparable to PM with Fab glycosylationwhile PM-N54Q showed a slightly better binding (FIG. 7A). Using HSC-4carcinoma cells that express TA-MUC1 at an intermediate level, thevariant PM-N54Q was clearly superior in binding to cellular TA-MUC1compared to PM while PM-N54D showed an inferior binding compared to PMwith Fab glycosylation (FIG. 7B).

Example 8: Evaluation of In Vitro Efficacy of PankoMab-ADC andPM-N54Q-ADC

8.1 Cell Lines

The human breast cancer cell line MDA-MB-468, the human pancreaticcancer cell line HPAC, and the human lung cancer cell line NCI-H441 wereused as TA-MUC1 medium to high-expressing cells. The human colorectalcancer cell line HCT-15 was used as TA-MUC1 negative cells. These celllines were purchased from ATCC. Each cell line was cultured inaccordance with an instruction manual. Expression level of TA-MUC1 oneach cancer cell line was confirmed by flow cytometry.

8.2 Evaluation of In Vitro Efficacy of PankoMab-ADC

MDA-MB-468 suspension was prepared to have a concentration of 1.25×10⁴cells/mL by using culture medium, and added to each well of a blackclear bottom 96-well plate at 80 uL/well (1000 cells/well). For blankwells, the medium alone was added to the wells at 80 uL/well (N=3). Allcells were incubated overnight in the appropriate condition forMDA-MB-468.

HCT-15 suspension was prepared to have a concentration of 3.1×10³cells/mL by using culture medium, suspension was added to each well of ablack clear bottom 96-well plate at 80 uL/well (250 cells/well). Forblank wells, the medium alone was added to the wells at 80 uL/well(N=3). All cells were incubated overnight in the appropriate conditionfor HCT-15.

On the next day, each naked PankoMab, control hIgG-ADC, and PankoMab-ADCwas 3-fold serially diluted with the each culture medium from 500 nM to0.2 nM. Twenty microliters of these diluted solutions were added to theappropriate wells (final concentration: 100 nM to 0.04 nM). For blankwells and untreated wells, 20 uL of the each culture medium alone wasadded to the wells. All plates were incubated for 6 days in theappropriate condition for each cell line.

After the incubations, the amount of ATP in each well was measured byusing a CellTiter-Glo Luminescent Cell Viability Assay (Promega).Luminescence was measured by a multilabel counter (ARVO X3, PerkinElmerJapan Co., Ltd.). This assay was performed in triplicate.

The cell viability of each sample was calculated by the followingequation:Cell viability (%)=100×(T−B)/(C−B)

T: the luminescence intensity of the test well

C: mean luminescence intensity of untreated wells

B: mean luminescence intensity of blank wells

8.3 Comparison of In Vitro Efficacy Between PankoMab-ADC and PM-N54Q-ADC

HPAC suspension was prepared to have a concentration of 1.25×10⁴cells/mL by using culture medium, and added to each well of a blackclear bottom 96-well plate at 80 uL/well (1000 cells/well). For blankwells, the medium alone was added to the wells at 80 uL/well (N=3). Allcells were incubated overnight in the appropriate condition for H PAC.

NCI-H441 suspension was prepared to have a concentration of 1.25×10⁴cells/mL by using culture medium, and added to each well of a blackclear bottom 96-well plate at 80 uL/well (1000 cells/well). For blankwells, the medium alone was added to the wells at 80 uL/well (N=3). Allcells were incubated overnight in the appropriate condition forNCI-H441.

On the next day, each naked PankoMab, naked PM-N54Q, hIgG-ADC,PankoMab-ADC, and PM-N54Q-ADC was 3-fold serially diluted with the eachculture medium from 500 nM to 0.2 nM. Twenty microliters of thesediluted solutions were added to the appropriate wells (finalconcentration: 100 nM to 0.04 nM). For blank wells and untreated wells,20 uL of the each culture medium alone was added to the wells. Allplates were incubated for 6 days in the appropriate condition for eachcell line.

After the incubations, the amount of ATP in each well was measured byusing a CellTiter-Glo Luminescent Cell Viability Assay (Promega).Luminescence was measured by a multilabel counter (ARVO X3, PerkinElmerJapan Co., Ltd.). This assay was performed in triplicate.

The cell viability of each sample was calculated by the followingequation:Cell viability (%)=100×(T−B)/(C−B)

T: the luminescence intensity of the test well

C: mean luminescence intensity of untreated wells

B: mean luminescence intensity of blank wells

Potency ratio of cytotoxic activity of PankoMab-ADC vs PM-N54Q-ADCagainst HPAC and NCI-H441, and their 95% CIs were calculated as post-hocanalysis using a 3-parameter logistic parallel-line analysis (commonslope) by using EXSUS ver.8.1 (CAC Croit, Tolyo, Japan) based on SASrelease 9.4 (SAS Institute Japan, Tokyo, Japan) (Emax: 100, Emin:estimate). The difference in the potency of cytotoxic activity wasconsidered to be significant if the 95% CI of potency ratio excluded 1.

Example 9: Evaluation of In Vivo Efficacy of PankoMab-ADC andPM-N54Q-ADC

9.1 Cell Lines

The human breast cancer cell line MDA-MB-468 and HCC70, the humanpancreatic cancer cell line HPAC, and the human lung cancer cell lineNCI-H441 were used as TA-MUC1 medium to high-expressing tumor cells. Thehuman colorectal cancer cell line HCT-15 was used as TA-MUC1 negativetumor cells. These cell lines were purchased from ATCC. The humanovarian cancer cell line OVCAR-5 was purchased from National CancerInstitute and used as TA-MUC1 low-expressing tumor cells. Each cell linewas cultured in accordance with an instruction manual. Expression levelof TA-MUC1 on each cancer cell line was confirmed by flow cytometry andIHC staining.

9.2 Evaluation of In Vivo Efficacy of PankoMab-ADC

MDA-MB-468 cells were suspended in Matrigel (BD), and 1×10⁷ cells weresubcutaneously transplanted to the right side of the body of each femalenude mice (Day 0), and the mice were randomly grouped on Day 20 (N=6).After grouping, each naked PankoMab, control hIgG-ADC, or PankoMab-ADCsolution was single dose administered intravenously at a dose of 3mg/kg. A vehicle (acetate buffer solution) administration group wasestablished as a control group. After administration, the tumor lengthand width of each mouse were measured with the digital caliper twice aweek for 21 days.

HCC70 cells were suspended in physiological saline (OtsukaPharmaceutical Factory, Inc.) and 1×10⁷ cells were subcutaneouslytransplanted to the right side of the body of each female nude mice (Day0), and the mice were randomly grouped on Day 19 (N=6). After grouping,each naked PankoMab, control hIgG-ADC, or PankoMab-ADC solution wassingle dose administered intravenously at a dose of 10 mg/kg. A vehicle(acetate buffer solution) administration group was established as acontrol group. After administration, the tumor length and width of eachmouse were measured with the digital caliper twice a week for 21 days.

The estimated tumor volume of each mouse was calculated by the followingequation:Estimated tumor volume (mm³)=½×length (mm)×width(mm)²

The tumor growth inhibition (TGI, %) of each group on the lastmeasurement day of vehicle treated groups was also calculated accordingto the following equation, and rounded to an integer.TGI (%)=(1−T/C)×100

T: the mean estimated tumor volume (mm³) of the naked PankoMab, controlhIgG-ADC, or PankoMab-ADC

C: the mean estimated tumor volume (mm³) of the vehicle treated group

In order to evaluate the anti-tumor efficacy of PankoMab-ADC, tumorvolumes of each mouse on the last measurement day of PankoMab-ADCtreated groups (MDA-MB-468: Day 41, HCC70: Day 40) were compared withthat of the control hIgG-ADC treated groups or that of naked PankoMabtreated group by Student t-test. All statistical analyses were performedas post-hoc analysis using SAS System Release 9.2 (SAS Institute Inc.).A P value of less than 0.05 was considered to be statisticallysignificant.

9.3 Comparison of In Vivo Efficacy of PankoMab-ADC and PM-N54Q-ADC

HPAC cells were suspended in physiological saline (Otsuka PharmaceuticalFactory, Inc.) and 3×10⁶ cells were subcutaneously transplanted to theright side of the body of each female nude mice (Day 0), and the micewere randomly grouped on Day 11 (N=6). After grouping, each nakedPM-N54Q, control hIgG-ADC, PankoMab-ADC, or PM-N54Q-ADC solution wassingle dose administered intravenously at a dose of 10 mg/kg. A vehicle(acetate buffer solution) administration group was established as acontrol group. After administration, the tumor length and width of eachmouse were measured with the digital caliper twice a week for 21 days.

NCI-H441 cells were suspended in Matrigel (BD), and 5×10⁶ cells weresubcutaneously transplanted to the right side of the body of each femalenude mice (Day 0), and the mice were randomly grouped on Day 7 (N=6).After grouping, each naked PankoMab or naked PM-N54Q solutions wassingle dose administered intravenously at a dose of 10 mg/kg, andcontrol hIgG-ADC, PankoMab-ADC, or PM-N54Q-ADC solution was single doseadministered intravenously at a dose of 3 mg/kg. A vehicle (acetatebuffer solution) administration group was established as a controlgroup. After administration, the tumor length and width of each mousewere measured with the digital caliper twice a week for 31 days.

OVCAR-5 cells were suspended in physiological saline (OtsukaPharmaceutical Factory, Inc.) and 5×10⁶ cells were subcutaneouslytransplanted to the right side of the body of each female nude mice (Day0), and the mice were randomly grouped on Day 12 (N=6). After grouping,naked PankoMab, naked PM-N54Q, control hIgG-ADC, PankoMab-ADC, orPM-N54Q-ADC solution was single dose administered intravenously at adose of 10 mg/kg. A vehicle (acetate buffer solution) administrationgroup was established as a control group. After administration, thetumor length and width of each mouse were measured with the digitalcaliper twice a week for 21 days.

HCT-15 cells were suspended in physiological saline (OtsukaPharmaceutical Factory, Inc.) and 5×10⁶ cells were subcutaneouslytransplanted to the right side of the body of each female nude mice (Day0), and the mice were randomly grouped on Day 10 (N=6). After grouping,control hIgG-ADC, PankoMab-ADC, or PM-N54Q-ADC solution was single doseadministered intravenously at a dose of 10 mg/kg. A vehicle (acetatebuffer solution) administration group was established as a controlgroup. After administration, the tumor length and width of each mousewere measured with the digital caliper twice a week for 21 days.

Tumor volume of each mouse was calculated by the following equation:Estimated tumor volume (mm³)=½×length (mm)×width (mm)²

The tumor growth inhibition (TGI, %) of each mouse on the lastmeasurement day of vehicle treated groups or the last day that allgroups are alive was also calculated according to the followingequation, and rounded to an integer.TGI (%)=(1−T/C)×100

T: the mean estimated tumor volume (mm³) of the naked PankoMab, nakedPM-N54Q, control hIgG-ADC, PankoMab-ADC or PM-N54Q-ADC

C: the mean estimated tumor volume (mm³) of the vehicle treated group

In order to evaluate the anti-tumor efficacy of each compound againstHPAC, NCI-H441, OVCAR-5, and HCT-15-bearing mice, tumor volumes of eachmouse on the last measurement day of control hIgG-ADC treated groups (HPAC: Day 32, NCI-H441: Day 38, HCT-15: Day 32) or the last day that allgroups are alive (OVCAR-5: Day 26) were compared with that of thecontrol hIgG-ADC treated groups by Dunnett's test. In addition, tumorvolumes of OVCAR-5-bearing nude mice on Day 33 were compared byStudent's t-test between PankoMab-ADC and PM-N54Q-ADC treated groups.All statistical analyses were performed as post-hoc analysis using SASSystem Release 9.2 (SAS Institute Inc.). A P value of less than 0.05 wasconsidered to be statistically significant.

Example 10: Results

10.1 Cytotoxic Activity of PankoMab-ADC Against TA-MUC1 Positive CancerCell Lines and Negative Cells In Vitro

To investigate whether PankoMab-ADC shows the target-dependent anddrug-dependent cytotoxic activity against human cancer cell lines, invitro efficacy of naked PankoMab, control hIgG-ADC, and PankoMab-ADCagainst the human breast cancer cells MDA-MB-468 (TA-MUC1 positive) andthe human colorectal cancer cells HCT-15 (TA-MUC1 negative) wasevaluated. As shown in FIG. 8 naked PankoMab and hIgG-ADC showed littleactivity against each cell line (IC₅₀>100 nM). Under these conditions,PankoMab-ADC exhibited dose-dependent cytotoxic activity against TA-MUC1positive cells MDA-MB-468 (FIG. 8A, IC₅₀<10 nM). But it didn't show theactivity against TA-MUC1 negative cells HCT-15 (FIG. 8B, IC₅₀>100 nM).Based on these results, it was concluded that PankoMab-ADC showstarget-dependent and drug-dependent cytotoxicactivity against TA-MUC1positive cancer cell lines in vitro.

10.2 Comparison of the In Vitro Cytotoxic Activity Between PankoMab-ADCand PM-N54Q-ADC Against TA-MUC1 Positive Cells In Vitro

To investigate whether improvement of antigen binding affinity maycontribute to enhancement of cytotoxic activity, in vitro efficacy ofPankoMab-ADC and PM-N54Q-ADC against the human pancreatic cancer cellline HPAC and the human lung cancer cell line NCI-H441 was evaluated.The cytotoxic activity of PM-N54Q-ADC against them was more than1.5-fold potent than that of PankoMab-ADC (FIG. 8C and FIG. 8D). Thepotency ratio of PM-N54Q-ADC to PankoMab-ADC against HPAC was 1.917(1.611−2.280, 95% CI), and that against NCI-H441 was 1.663 (1.495 to1.849, 95% CI at EC50). These data demonstrated that cytotoxic activityof PM-N54Q-ADC is significantly more potent than that of PankoMab-ADC.These results suggest that improvement of antigen binding affinity ofPankoMab-ADC may contribute to significant enhancement of cell killingactivity.

10.3 Anti-Tumor Efficacy of PankoMab-ADC Against TA-MUC1 Positive Tumor

To investigate whether PankoMab-ADC shows not only in vitro but also invivo efficacy, anti-tumor efficacy of naked PankoMab, control hIgG-ADC,and PankoMab-ADC against MDA-MB-468-bearing mice was evaluated. As shownin FIG. 9 , naked PankoMab and control IgG-ADC (3 mg/kg, singleadministration) didn't show anti-tumor efficacy (both of TGIs were −18%on Day 41). By contrast, PankoMab-ADC (3 mg/kg, single administration)remarkably inhibited the tumor growth (TGI was 97% on Day 41). Moreover,it showed significant anti-tumor efficacy compared to control hIgG-ADCand naked PankoMab (both of P<0.001 on Day 41). In terms of body weightchange, any body weight loss caused by drug treatment was not observedin all drug-treatment groups.

Anti-tumor efficacy of naked PankoMab, control hIgG-ADC, andPankoMab-ADC against HCC70-bearing mice was also evaluated. As shown inFIG. 10 , naked PankoMab and control hIgG-ADC (10 mg/kg, singleadministration) showed weak anti-tumor efficacy against these xenograftmodels (TGI was 10% and 29% on Day 40, respectively). By contrast,PankoMab-ADC (10 mg/kg, single administration) remarkably inhibited thetumor growth (TGI was 95% on Day 40). Moreover, it showed statisticallysignificant anti-tumor efficacy compared to control hIgG-ADC (both ofP<0.001 on Day 40). In terms of body weight change, any body weight losscaused by drug treatment was not observed in all drug-treatment groups.These results suggest that PankoMab-ADC has strong anti-tumor efficacyand it showed target-dependent and drug-dependent anti-tumor efficacyagainst various TA-MUC1 positive xenograft models.

10.4 Comparison of the Anti-Tumor Efficacy Between PankoMab-ADC andPM-N54Q-ADC Against TA-MUC1 Positive Tumor In Vivo

To investigate whether PM-N54Q-ADC has equal to or greater anti-tumorefficacy against TA-MUC1 positive tumor cells than PankoMab-ADC,anti-tumor efficacy of PankoMab-ADC and PM-N54Q-ADC against varioustypes of TA-MUC1 positive tumor cells was compared.

At first, we evaluated the in vivo efficacy against HPAC and NCI-H441tumor cells with medium to high TA-MUC1 expression. As shown in FIG. 11, naked PM-N54Q and control hIgG-ADC (10 mg/kg, single administration)showed weak anti-tumor efficacy against HPAC-bearing mice (TGI was 27%and 18% on Day 32, respectively). By contrast, PankoMab-ADC andPM-N54Q-ADC (10 mg/kg, single administration) remarkably inhibited thetumor growth (both of TGIs were 93% on Day 32). Moreover, PankoMab-ADCand PM-N54Q-ADC (10 mg/kg, single administration) showed statisticallysignificant anti-tumor efficacy compared to control hIgG-ADC (both ofP<0.001 on Day 32). In terms of body weight change, any body weight losscaused by drug treatment was not observed in all drug-treatment groups.

As shown in FIG. 12 , naked PankoMab and naked PM-N54Q (10 mg/kg, singleadministration) showed weak anti-tumor efficacy against NCI-H441-bearingmice (TGI was 8% and 12% on Day 38, respectively). Although controlhIgG-ADC (3 mg/kg, single administration) treated group showedanti-tumor efficacy for two weeks after administration, tumor regrowthwas observed after day 21 (TGI was 71% on Day 38). By contrast,PankoMab-ADC and PM-N54Q-ADC (3 mg/kg, single administration) remarkablyinhibited the tumor growth (both of TGI was 99% on Day 38). Moreover,PankoMab-ADC and PM-N54Q-ADC showed statistically significant anti-tumorefficacy compared to control hIgG-ADC (P<0.001 on Day 38, respectively).In terms of body weight change, any body weight loss caused by drugtreatment was not observed in all drug-treatment groups.

Next, we evaluated the in vivo efficacy against OVCAR-5 tumor cells inwhich TA-MUC1 low expression. As shown in FIG. 13 , naked PankoMab,PM-N54Q and control hIgG-ADC (10 mg/kg, single administration) showedlittle anti-tumor efficacy against OVCAR5-bearing mice (TGI was 1%, 11%and 3% on Day 26, respectively). In this model, anti-tumor efficacy ofPankoMab-ADC (10 mg/kg, single administration) was limited (TGI was 37%on Day 26), but PM-N54Q-ADC (10 mg/kg, single administration) showedstrong anti-tumor efficacy (TGI was 73% on Day 26). Moreover,PankoMab-ADC and PM-N54Q-ADC showed statistically significant anti-tumorefficacy compared to control hIgG-ADC (P=0.01 and P<0.001 on Day 26,respectively). In addition, PM-N54Q-ADC showed statistically significantanti-tumor efficacy compared to PankoMab-ADC (P<0.001 on Day 26). Interms of body weight change, any body weight loss caused by drugtreatment was not observed in all drug-treatment groups.

Finally, we evaluated the in vivo efficacy against HCT-15 tumor cellswhich TA-MUC1 negative.

As shown in FIG. 14 , naked PankoMab and PM-N54Q (10 mg/kg, singleadministration) showed little anti-tumor efficacy against this model(TGI was 7%, 4% on Day 32, respectively). Moreover, PankoMab-ADC,PM-N54Q-ADC and control hIgG-ADC also showed little anti-tumor efficacyagainst this model (TGI was 15%, 22%, and 26% on Day 32, respectively).

Based on these results, it was concluded that the anti-tumor efficacy ofPankoMab-ADC and PM-N54Q-ADC is target-dependent and drug-dependent.And, improvement of antigen binding affinity may contribute toenhancement of anti-tumor efficacy against TA-MUC1 positive tumor cells.

Identification of the Deposited Biological Material

The cell lines DSM ACC 2806, DSM ACC 2807 and DSM ACC 2856 weredeposited at the DSMZ—Deutsche Sammlung von Mikroorganismen andZellkulturen GmbH, Inhoffenstraße 7B, 38124 Braunschweig (DE) byGlycotope GmbH, Robert-Rössle-Str. 10, 13125 Berlin (DE) on the datesindicated in the following table.

Name of the Accession Date of Cell Line Number Depositor DepositionNM-H9D8 DSM ACC 2806 Glycotope GmbH Sep. 15, 2006 NM-H9D8-E6 DSM ACC2807 Glycotope GmbH Oct. 5, 2006 NM-H9D8-E6Q12 DSM ACC 2856 GlycotopeGmbH Aug. 8, 2007

The invention claimed is:
 1. A conjugate, which is represented thefollowing formula:

wherein AB represents an antibody, the antibody comprising a heavy chainvariable region having the amino acid sequence of SEQ ID NO: 10 and alight chain variable region having the amino acid sequence of SEQ ID NO:12, y represents an average number of units of the drug-linker structureconjugated to the antibody per itself, and the antibody is conjugated toa drug linker represented by the above formula by a thioether bond.
 2. Aconjugate, which is represented by the following formula,

wherein AB represents an antibody, the antibody comprising a heavy chainhaving the amino acid sequence of SEQ ID NO: 22 or a variant thereof inwhich one amino acid has been removed from the C-terminus and a lightchain having the amino acid sequence of SEQ ID NO: 16, y represents anaverage number of units of the drug-linker structure conjugated to theantibody per itself, and the antibody is conjugated to a drug linkerrepresented by the above formula by a thioether bond.
 3. Apharmaceutical composition comprising the conjugate of claim
 2. 4. Theconjugate according to claim 2, wherein the antibody comprises adeletion or lack of one or two amino acid(s) in the carboxyl terminus ofthe heavy chain.
 5. The conjugate according to claim 2, wherein theantibody comprises two heavy chains, both of which lack onecarboxyl-terminal amino acid residue.
 6. The conjugate according toclaim 2, wherein the average number of units of the drug-linkerstructure y conjugated per antibody is in a range of from 2 to
 8. 7. Theconjugate according to claim 2, wherein the antibody comprises one ormore modifications selected from the group consisting of defucosylation,reduced fucose, N-linked glycosylation, O-linked glycosylation,N-terminal processing, C-terminal processing, deamidation, isomerizationof aspartic acid, oxidation of methionine, the substitutions of twoleucine (L) residues to alanine (A) at position 234 and 235 of the heavychain (LALA), amidation of a proline residue, and a deletion or lack ofone or two amino acids at the carboxyl terminus.
 8. The conjugateaccording to claim 2, wherein the average number of units of thedrug-linker structure y conjugated per antibody is in a range of from 1to
 10. 9. The conjugate according to claim 2, wherein the average numberof units of the drug-linker structure y conjugated per antibody is in arange of from 3 to
 8. 10. The conjugate according to claim 2, whereinthe average number of units of the drug-linker structure y conjugatedper antibody is in a range of from 7 to
 8. 11. The conjugate accordingto claim 2, wherein the average number of units of the drug-linkerstructure y conjugated per antibody is in a range of from 7.5 to
 8. 12.The conjugate according to claim 2, wherein the number of units of thedrug-linker structure conjugated per antibody molecule is
 8. 13. Theconjugate according to claim 1, wherein the antibody comprises one ormore modifications selected from the group consisting of defucosylation,reduced fucose, N-linked glycosylation, O-linked glycosylation,N-terminal processing, C-terminal processing, deamidation, isomerizationof aspartic acid, oxidation of methionine, the substitutions of twoleucine (L) residues to alanine (A) at position 234 and 235 of the heavychain (LALA), amidation of a proline residue, and a deletion or lack ofone or two amino acids at the carboxyl terminus.
 14. The conjugateaccording to claim 1, wherein the antibody comprises a deletion or lackof one or two amino acid(s) in the carboxyl terminus of the heavy chain.15. The conjugate according to claim 1, wherein the antibody comprisestwo heavy chains, both of which lack one carboxyl-terminal amino acidresidue.
 16. The conjugate according to claim 1, wherein the averagenumber of units of the drug-linker structure y conjugated per antibodyis in a range of from 1 to
 10. 17. The conjugate according to claim 1,wherein the average number of units of the drug-linker structure yconjugated per antibody is in a range of from 2 to
 8. 18. The conjugateaccording to claim 1, wherein the average number of units of thedrug-linker structure y conjugated per antibody is in a range of from 3to
 8. 19. The conjugate according to claim 1, wherein the average numberof units of the drug-linker structure y conjugated per antibody is in arange of from 7 to
 8. 20. The conjugate according to claim 1, whereinthe average number of units of the drug-linker structure y conjugatedper antibody is in a range of from 7.5 to
 8. 21. The conjugate accordingto claim 1, wherein the number of units of the drug-linker structureconjugated per antibody molecule is
 8. 22. A pharmaceutical compositioncomprising the conjugate of claim 1.